Defining the role of IL-15 trans-presentation by distinct cell-types during the development and homeostasis of Natural Killer and invariant Natural Killer T cells

The immuno-regulatory functions displayed by NK and iNKT cells have highlighted their importance as key lymphocytes involved in innate and adaptive immunity. Therefore, understanding the dynamics influencing the generation of NK and iNKT cells is extremely important. IL-15 has been shown to provide a critical signal throughout the development and homeostasis of NK and iNKT cells; however, the cellular source of IL-15 has remained unclear. In this investigation, I provide evidence that the cell-type providing IL-15 to NK and iNKT cells via trans-presentation is determined by the tissue site and the maturation status of NK and iNKT cells. For NK cells, I revealed the non-hematopoietic compartment provides IL-15 to NK cells in the early stages of development while hematopoietic cells were crucial for the generation and maintenance of mature NK cells. Regarding iNKT cells in the thymus, IL-15 trans-presentation by non-hematopoietic cells was crucial for the survival of mature iNKT cells. In the liver, both hematopoietic and non-hematopoietic compartments provided IL-15 to both immature and mature iNKT cells. This IL-15 signal helped mediate the survival and proliferation of both NK and iNKT cells as well as induce the functional maturation of mature iNKT cells via enhanced T-bet expression. In conclusion, my work illustrates an important notion that the immunological niche of NK and iNKT cells is tightly regulated and that this regulation is meticulously influenced by the tissue microenvironment.

Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid.

Linezolid, which targets the ribosome, is a new synthetic antibiotic that is used for treatment of infections caused by Gram-positive pathogens. Clinical resistance to linezolid, so far, has been developing only slowly and has involved exclusively target site mutations. We have discovered that linezolid resistance in a methicillin-resistant Staphylococcus aureus hospital strain from Colombia is determined by the presence of the cfr gene whose product, Cfr methyltransferase, modifies adenosine at position 2503 in 23S rRNA in the large ribosomal subunit. The molecular model of the linezolid-ribosome complex reveals localization of A2503 within the drug binding site. The natural function of cfr likely involves protection against natural antibiotics whose site of action overlaps that of linezolid. In the chromosome of the clinical strain, cfr is linked to ermB, a gene responsible for dimethylation of A2058 in 23S rRNA. Coexpression of these two genes confers resistance to all the clinically relevant antibiotics that target the large ribosomal subunit. The association of the ermB/cfr operon with transposon and plasmid genetic elements indicates its possible mobile nature. This is the first example of clinical resistance to the synthetic drug linezolid which involves a natural resistance gene with the capability of disseminating among Gram-positive pathogenic strains.

Multiple site phosphorylation of tyrosine hydroxylase: A potential role for protein kinase C in the regulation of catecholamine synthesis

Tyrosine hydroxylase (TH), the initial and rate limiting enzyme in the catecholaminergic biosynthetic pathway, is phosphorylated on multiple serine residues by multiple protein kinases. Although it has been demonstrated that many protein kinases are capable of phosphorylating and activating TH in vitro, it is less clear which protein kinases participate in the physiological regulation of catecholamine synthesis in situ. These studies were designed to determine if protein kinase C (PK-C) plays such a regulatory role.^ Stimulation of intact bovine adrenal chromaffin cells with phorbol esters results in stimulation of catecholamine synthesis, tyrosine hydroxylase phosphorylation and activation. These responses are both time and concentration dependent, and are specific for those phorbol ester analogues which activate PK-C. RP-HPLC analysis of TH tryptic phosphopeptides indicate that PK-C phosphorylates TH on three putative sites. One of these (pepetide 6) is the same as that phosphorylated by both cAMP-dependent protein kinase (PK-A) and calcium/calmodulin-dependent protein kinase (CaM-K). However, two of these sites (peptides 4 and 7) are unique, and, to date, have not been shown to be phosphorylated by any other protein kinase. These peptides correspond to those which are phosphorylated with a slow time course in response to stimulation of chromaffin cells with the natural agonist acetylcholine. The activation of TH produced by PK-C is most closely correlated with the phosphorylation of peptide 6. But, as evident from pH profiles of tyrosine hydroxylase activity, phosphorylation of peptides 4 and 7 affect the expression of the activation produced by phosphorylation of peptide 6.^ These data support a role for PK-C in the control of TH activity, and suggest a two stage model for the physiological regulation of catecholamine synthesis by phosphorylation in response to cholinergic stimulation. An initial fast response, which appears to be mediated by CaM-K, and a slower, sustained response which appears to be mediated by PK-C. In addition, the multiple site phosphorylation of TH provides a mechanism whereby the regulation of catecholamine synthesis appears to be under the control of multiple protein kinases, and allows for the convergence of multiple, diverse physiological and biochemical signals. ^

Climatic drivers of hourly to yearly tree radius variations along a 6°C natural warming gradient

Climate affects the timing, rate and dynamics of tree growth, over time scales ranging from seconds to centuries. Monitoring how a tree's stem radius varies over these time scales can provide insight into intra-annual stem dynamics and improve our understanding of climate impacts on tree physiology and growth processes. Here, we quantify the response of radial conifer stem size to environmental fluctuations via a novel assessment of tree circadian cycles. We analyze four years of sub-hourly data collected from 56 larch and spruce trees growing along a natural temperature gradient of ∼6 °C in the central Swiss Alps. During the growing season, tree stem diameters were greatest at mid-morning and smallest in the late evening, reflecting the daily cycle of water uptake and loss. Along the gradient, amplitudes calculated from the stem radius cycle were ∼50% smaller at the upper site (∼2200 m a.s.l.) relative to the lower site (∼800 m a.s.l.). We show changes in precipitation, temperature and cloud cover have a substantial effect on typical growing season diurnal cycles; amplitudes were nine times smaller on rainy days (>10 mm), and daily amplitudes are approximately 40% larger when the mean daily temperature is 15–20 °C than when it is 5–10 °C. We find that over the growing season in the sub-alpine forests, spruce show greater daily stem water movement than larch. However, under projected future warming, larch could experience up to 50% greater stem water use, which may severely affect future growth on already dry sites. Our data further indicate that because of the confounding influences of radial growth and short-term water dynamics on stem size, conventional methodology probably overstates the effect of water-linked meteorological variables (i.e. precipitation and relative humidity) on intra-annual tree growth. We suggest future studies use intra-seasonal measurements of cell development and consider whether climatic factors produce reversible changes in stem diameter. These study design elements may help researchers more accurately quantify and attribute changes in forest productivity in response to future warming.

Matrix pore water in low-permeable crystalline bedrock: An archive for the palaeohydrogeological evolution of the Olkiluoto Investigation Site

Matrix pore water in the connected inter- and intragranular pore space of low-permeable crystalline bedrock interacts with flowing fracture groundwater predominately by diffusion. Based on the slow exchange between the two water reservoirs, matrix pore water acts as an archive of past changes in fracture groundwater compositions and thus of the palaeohydrological history of a site. Matrix pore water of crystalline bedrock from the Olkiluoto investigation site (SW Finland) was characterised using the stable water isotopes (δ18O, δ2H), combined with the concentrations of dissolved chloride and bromide as natural tracers. The comparison of tracer concentrations in pore water and present-day fracture groundwater suggest for the pore water the presence of old, dilute meteoric water components that infiltrated into the fractures during various warm climate stages. These different meteoric components can be discerned based on the diffusion distance between the two reservoirs and be brought into context with the palaeohydrological evolution of the site.

Contrasting vulnerability and resilience to drought-induced decline of densely planted vs. natural rear-edge Pinus nigra forests

The southernmost European natural and planted pine forests are among the most vulnerable areas to warming-induced drought decline. Both drought stress and management factors (e.g., stand origin or reduced thinning) may induce decline by reducing the water available to trees but their relative importances have not been properly assessed. The role of stand origin - densely planted vs. naturally regenerated stands - as a decline driver can be assessed by comparing the growth and vigor responses to drought of similar natural vs. planted stands. Here, we compare these responses in natural and planted Black pine (Pinus nigra) stands located in southern Spain. We analyze how environmental factors - climatic (temperature and precipitation anomalies) and site conditions - and biotic factors - stand structure (age, tree size, density) and defoliation by the pine processionary moth - drive radial growth and crown condition at stand and tree levels. We also assess the climatic trends in the study area over the last 60 years. We use dendrochronology, linear mixed-effects models of basal area increment and structural equation models to determine how natural and planted stands respond to drought and current competition intensity. We observed that a temperature rise and a decrease in precipitation during the growing period led to increasing drought stress during the late 20th century. Trees from planted stands experienced stronger growth reductions and displayed more severe crown defoliation after severe droughts than those from natural stands. High stand density negatively drove growth and enhanced crown dieback, particularly in planted stands. Also pine processionary moth defoliation was more severe in the growth of natural than in planted stands but affected tree crown condition similarly in both stand types. In response to drought, sharp growth reduction and widespread defoliation of planted Mediterranean pine stands indicate that they are more vulnerable and less resilient to drought stress than natural stands. To mitigate forest decline of planted stands in xeric areas such as the Mediterranean Basin, less dense and more diverse stands should be created through selective thinning or by selecting species or provenances that are more drought tolerant. (C) 2013 Elsevier B.V. All rights reserved.

Ecosystem impacts of feral rabbits on World Heritage sub-Antarctic Macquarie Island: A palaeoecological perspective

The introduction and establishment of non-indigenous species through human activities often poses a major threat to natural biodiversity. In many parts of the world management efforts are therefore focused on their eradication. The environment of World Heritage sub-Antarctic Macquarie Island has been severely damaged by non-indigenous species including rabbits, rats and mice, introduced from the late AD 1800s. An extensive eradication programme is now underway which aims to remove all rabbits and rodents. To provide a long-term context for assessing the Island's pre-invasion state, invasion impacts, and to provide a baseline for monitoring its recovery, we undertook a palaeoecological study using proxies in a lake sediment core. Sedimentological and diatom analyses revealed an unproductive catchment and lake environment persisted for ca. 7100 years prior to the introduction of the invasive species. After ca. AD 1898, unprecedented and statistically significant environmental changes occurred. Lake sediment accumulation rates increased >100 times due to enhanced catchment inputs and within-lake production. Total carbon and total nitrogen contents of the sediments increased by a factor of four. The diatom flora became dominated by two previously rare species. The results strongly suggest a causal link between the anthropogenic introduction of rabbits and the changes identified in the lake sediments. This study provides an example of how palaeoecology may be used to determine baseline conditions prior to the introduction of non-indigenous species, quantify the timing and extent of changes, and help monitor the recovery of the ecosystem and natural biodiversity following successful non-indigenous species eradication programmes.

Synthesis and testing of photoaffinity probes for the site-selectivechemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Natural antisense transcripts of Trifolium repens dehydrins

The recently described complex nature of some dehydrin-coding sequences in Trifolium repens could explain the considerable variability among transcripts originating from a single gene.1 For some of the sequences the existence of natural antisense transcripts (NAT s), which could form sense-antisense (SAS) pairs, was predicted. The present study demonstrates that cis-natural antisense transcripts of 2 dehydrin types (YnKn and YnSKn) accumulate in white clover plants subjected to treatments with polyethylene glycol (PEG), abscisic acid (ABA), and high salt concentration. The isolated YnKn cis-NAT s mapped to sequence site enriched in alternative start codons. Some of the sense-antisense pairs exhibited inverse expression with differing profiles which depended on the applied stress. A natural antisense transcript coding for an ABC F family protein (a trans-NAT) which shares short sequence homology with YnSKn dehydrin was identified in plants subjected to salt stress. Forthcoming experiments will evaluate the impact of NAT s on transcript abundances, elucidating the role of transcriptional and post-transcriptional interferences in the regulation of dehydrin levels under various abiotic stresses.

The integrity of the G2421-C2395 base pair in the ribosomal E-site is crucial for protein synthesis.

During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.

A high-affinity natural autoantibody from human cord blood defines a physiologically relevant epitope on the FcepsilonRIalpha.

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the alpha domain of the high-affinity IgE receptor (FcepsilonRIalpha) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcepsilonRIalpha Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcepsilonRIalpha had the same sequence. This single IgM Ab, named CBMalpha8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcepsilonRIalpha recognized by both CBMalpha8 and the previously isolated anti-FcepsilonRIalpha Abs from autoimmune and healthy donors. This common epitope on FcepsilonRIalpha coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMalpha8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcepsilonRIalpha Abs are rendered less likely to compete with IgE for binding to FcepsilonRIalpha.

A thermodynamic model for di-trioctahedral chlorite from experimental and natural data in the system MgO-FeO-Al2O3-SiO2-H2O. Applications to P-T sections and geothermometry

We present a new thermodynamic activity-composition model for di-trioctahedral chlorite in the system FeO–MgO–Al2O3–SiO2–H2O that is based on the Holland–Powell internally consistent thermodynamic data set. The model is formulated in terms of four linearly independent end-members, which are amesite, clinochlore, daphnite and sudoite. These account for the most important crystal-chemical substitutions in chlorite, the Fe–Mg, Tschermak and di-trioctahedral substitution. The ideal part of end-member activities is modeled with a mixing-on-site formalism, and non-ideality is described by a macroscopic symmetric (regular) formalism. The symmetric interaction parameters were calibrated using a set of 271 published chlorite analyses for which robust independent temperature estimates are available. In addition, adjustment of the standard state thermodynamic properties of sudoite was required to accurately reproduce experimental brackets involving sudoite. This new model was tested by calculating representative P–T sections for metasediments at low temperatures (<400 °C), in particular sudoite and chlorite bearing metapelites from Crete. Comparison between the calculated mineral assemblages and field data shows that the new model is able to predict the coexistence of chlorite and sudoite at low metamorphic temperatures. The predicted lower limit of the chloritoid stability field is also in better agreement with petrological observations. For practical applications to metamorphic and hydrothermal environments, two new semi-empirical chlorite geothermometers named Chl(1) and Chl(2) were calibrated based on the chlorite + quartz + water equilibrium (2 clinochlore + 3 sudoite = 4 amesite + 4 H2O + 7 quartz). The Chl(1) thermometer requires knowledge of the (Fe3+/ΣFe) ratio in chlorite and predicts correct temperatures for a range of redox conditions. The Chl(2) geothermometer which assumes that all iron in chlorite is ferrous has been applied to partially recrystallized detrital chlorite from the Zone houillère in the French Western Alps.

3CAPS – a structural AP-site analogue as a tool to investigate DNA base excision repair

Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP-sites. With its 3′-phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′-deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.

Digital Vertical Aerial Camera System for High-resolution Site Inspections in Conservation Easment Monitoring

A three-level satellite to ground monitoring scheme for conservation easement monitoring has been implemented in which high-resolution imagery serves as an intermediate step for inspecting high priority sites. A digital vertical aerial camera system was developed to fulfill the need for an economical source of imagery for this intermediate step. A method for attaching the camera system to small aircraft was designed, and the camera system was calibrated and tested. To ensure that the images obtained were of suitable quality for use in Level 2 inspections, rectified imagery was required to provide positional accuracy of 5 meters or less to be comparable to current commercially available high-resolution satellite imagery. Focal length calibration was performed to discover the infinity focal length at two lens settings (24mm and 35mm) with a precision of O.1mm. Known focal length is required for creation of navigation points representing locations to be photographed (waypoints). Photographing an object of known size at distances on a test range allowed estimates of focal lengths of 25.lmm and 35.4mm for the 24mm and 35mm lens settings, respectively. Constants required for distortion removal procedures were obtained using analytical plumb-line calibration procedures for both lens settings, with mild distortion at the 24mm setting and virtually no distortion found at the 35mm setting. The system was designed to operate in a series of stages: mission planning, mission execution, and post-mission processing. During mission planning, waypoints were created using custom tools in geographic information system (GIs) software. During mission execution, the camera is connected to a laptop computer with a global positioning system (GPS) receiver attached. Customized mobile GIs software accepts position information from the GPS receiver, provides information for navigation, and automatically triggers the camera upon reaching the desired location. Post-mission processing (rectification) of imagery for removal of lens distortion effects, correction of imagery for horizontal displacement due to terrain variations (relief displacement), and relating the images to ground coordinates were performed with no more than a second-order polynomial warping function. Accuracy testing was performed to verify the positional accuracy capabilities of the system in an ideal-case scenario as well as a real-world case. Using many welldistributed and highly accurate control points on flat terrain, the rectified images yielded median positional accuracy of 0.3 meters. Imagery captured over commercial forestland with varying terrain in eastern Maine, rectified to digital orthophoto quadrangles, yielded median positional accuracies of 2.3 meters with accuracies of 3.1 meters or better in 75 percent of measurements made. These accuracies were well within performance requirements. The images from the digital camera system are of high quality, displaying significant detail at common flying heights. At common flying heights the ground resolution of the camera system ranges between 0.07 meters and 0.67 meters per pixel, satisfying the requirement that imagery be of comparable resolution to current highresolution satellite imagery. Due to the high resolution of the imagery, the positional accuracy attainable, and the convenience with which it is operated, the digital aerial camera system developed is a potentially cost-effective solution for use in the intermediate step of a satellite to ground conservation easement monitoring scheme.

THE NATURAL BACKGROUND GAMMA RADIATION EXPOSURE IN THE METROPOLITAN AREA OF THE VALLEY OF MEXICO

Measurements of the natural background radiation have been made at numerous places throughout the world. Very little work in this field has been done in developing countries. In Mexico the natural radiation to which the population is exposed has not been assessed. This dissertation represents a pioneer study in this environmental area. The radiation exposure which occupants within buildings receive as a result of naturally occurring radionuclides present in construction materials is the principal focus.^ Data were collected between August 1979 and November 1980. Continuous monitoring was done with TLDs placed on site for periods of 3 to 6 months. The instrumentation used for "real-time" measurements was a portable NaI (Tl) scintillation detector. In addition, radiometric measurements were performed on construction materials commonly used in Mexican homes.^ Based on TLD readings taken within 75 dwellings, the typical indoor exposure for a resident of the study area is 9.2 (mu)Rh('-1). The average reading of the 152 indoor scintillometer surveys was 9.5 (mu)Rh('-1), the outdoor reading 7.5 (mu)Rh('-1). Results of one-way and multi-way analyses of the exposure data to determine the effect due to building materials type, geologic subsoil, age of dwelling, and elevation are also presented. The results of 152 indoor scintillometer surveys are described. ^