Anti-CD20 Immunoglobulin G Radiolabeling with a 99mTc-Tricarbonyl Core: In Vitro and In Vivo Evaluations

In recent years, the diagnostic and therapeutic uses of radioisotopes have shown significant progress. Immunoglobulin (Ig) appears to be a promising tracer, particularly due to its ability to target selected antigens. The main objective of this study is to optimize and assess an Ig radiolabeling method with Technetium 99m (^99mTc), an attractive radioelement used widely for diagnostic imaging. Monoclonal anti-CD20 IgG was retained to study in vitro and in vivo radiolabeling impact. After IgG derivatization with 2-iminothiolane, IgG-SH was radiolabeled by an indirect method, using a ^99mTc-tricarbonyl core. Radiolabeling stability was evaluated over 24h by thin-layer chromatography. IgG integrity was checked by sodium dodecyl sulfate—polyacrylamide gel electrophoresis coupled with Western blot and autoradiography. The radiolabeled Ig’s immunoaffinity was assessed in vitro by a radioimmunoassay method and binding experiments with cells (EL4-hCD20 and EL4-WT). Biodistribution studies were performed in normal BALB/c mice. Tumor uptake was assessed in mice bearing EL4-hCD20 and EL4-WT subcutaneous xenografts. With optimized method, high radiolabeling yields were obtained (95.9 ± 3.5%). ^99mTc-IgG-SH was stable in phosphate-buffered saline (4°C and 25°C) and in serum (37°C), even if important sensitivity to transchelation was observed. IgG was not degraded by derivatization and radiolabeling, as shown by Western blot and autoradiography results. ^99mTc-anti-CD20 IgG-SH immunoaffinity was estimated with Kd = 35 nM by both methods. In vivo biodistribution studies for 48h showed significant accumulation of radioactivity in plasma, liver, spleen, lungs and kidneys. Planar scintigraphy of mice bearing tumors showed a significant uptake of ^99mTc-anti-CD20 IgG-SH in CD20⁺ tumor versus CD20^- tumor. Radiolabeling of derivatized IgG with ^99mTc-tricarbonyl was effective, stable and required few antibody amounts. This attractive radiolabeling method is “antibody safe” and preserves Ig affinity for antigen, as shown by both in vitro and in vivo experiments. This method could easily be used with noncommercial IgG or other antibody isotypes.     

Disentangling the paradox of insect phenology: are temporal trends reflecting the response to warming?

The strength and direction of phenological responses to changes in climate have been shown to vary significantly both among species and among populations of a species, with the overall patterns not fully resolved. Here, we studied the temporal and spatial variability associated with the response of several insect species to recent global warming. We use hierarchical models within a model comparison framework to analyze phenological data gathered over 40 years by the Japan Meteorological Agency on the emergence dates of 14 insect species at sites across Japan. Contrary to what has been predicted with global warming, temporal trends of annual emergence showed a later emergence day for some species and sites over time, even though temperatures are warming. However, when emergence data were analyzed as a function of temperature and precipitation, the overall response pointed out an earlier emergence day with warmer conditions. The apparent contradiction between the response to temperature and trends over time indicates that other factors, such as declining populations, may be affecting the date phenological events are being recorded. Overall, the responses by insects were weaker than those found for plants in previous work over the same time period in these ecosystems, suggesting the potential for ecological mismatches with deleterious effects for both suites of species. And although temperature may be the major driver of species phenology, we should be cautious when analyzing phenological datasets as many other factors may also be contributing to the variability in phenology.     

Variable selection and accurate predictions in habitat modelling: a shrinkage approach

Habitat modelling is increasingly relevant in biodiversity and conservation studies. A typical application is to predict potential zones of specific conservation interest. With many environmental covariates, a large number of models can be investigated but multi‐model inference may become impractical. Shrinkage regression overcomes this issue by dealing with the identification and accurate estimation of effect size for prediction. In a Bayesian framework we investigated the use of a shrinkage prior, the Horseshoe, for variable selection in spatial generalized linear models (GLM). As study cases, we considered 5 datasets on small pelagic fish abundance in the Gulf of Lion (Mediterranean Sea, France) and 9 environmental inputs. We compared the predictive performances of a simple kriging model, a full spatial GLM model with independent normal priors for regression coefficients, a full spatial GLM model with a Horseshoe prior for regression coefficients and 2 zero‐inflated models (spatial and non‐spatial) with a Horseshoe prior. Predictive performances were evaluated by cross‐validation on a hold‐out subset of the data: models with a Horseshoe prior performed best, and the full model with independent normal priors worst. With an increasing number of inputs, extrapolation quickly became pervasive as we tried to predict from novel combinations of covariate values. By shrinking regression coefficients with a Horseshoe prior, only one model needed to be fitted to the data in order to obtain reasonable and accurate predictions, including extrapolations.     

A rapid mini-prep method for isolation of ectomycorrhizal DNA from Tuber species

A rapid procedure has been developed to isolate DNA from the ectomycorrhizae of Tuber spp. for use in PCR experiments. The method described is fast and sensitive and can overcome the amplification problems that can arise in the presence of inhibitors. For this reason it can be used to type ectomycorrhizae even starting from a single root tip and make mycorrhizae identification much more rapid.     

Synthesis,characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels

Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In this report, we determine the three-dimensional structure of HgTX(1) using NMR spectroscopy (PDB-code: 1HLY). HgTX(1) was found to possess a structure similar to previously characterized K+ channel toxins (e.g. margatoxin) consisting of a three-stranded antiparallel beta-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 3(10) helix and part alpha helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interaction with the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX(1). On the basis of previous studies (see above) and our NMR data, a HgTX(1) mutant (HgTX(1)-A19C) was engineered, expressed, and purified. HgTX(1)-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-, and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX(1)-A19C in radioligand binding studies indicated that these hongotoxin(1) analogues retain high-affinity for voltage-gated K+ channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin(1) analogues were used to investigate the localization of K+ channels in brain sections. The distribution of toxin binding closely follows the distribution of K(v)1.2 immunoreactivity with the highest expression levels in the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescently labeled HgTX(1) analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.     

Nucleolar Organizer ultrastructure in Allium cepa

The Nuoleolar Organizer Region (NOR) was studied in Allium cepa. The cells analyzed were: meristematic root tip cells, cells from the layers of the anther (exothecium, intermediate layer cells, endothecium and tapetum) and meiocytes from preleptotene to diptotene. Conventional electron microscope techniques and serial section were used in the study. Ultracytochemical tests, using preferential techniques for nucleic acids were also applied. The results show that: a) Although the NOR may have a very variable form in the different cells analyzed, in each case its fine structure is similar, being constituted of chromatin differentiated in high and low density zones. b) In each case, its cytochemical characteristics are similar; highly positively to EDTA (preferential stain for RNP) being observed in the low density zones. As scattered chromatin these would constitute the active regions of the NOR. c) In certain cases a clear relationship is seen between NOR volume and the size of the nucleolus. d) The chromatin associated to the NOR always appears as very dense zones. e) The NOR-Nucleolus relationships are established exclusively with the latter's fibrillar pars. f) The nucleolar segregation phenomenon takes place sometimes as a result of a NOR contraction and in other cases depends on the position of the NOR in relation to the nucleolus. g) In preleptotene, the NOR shows its characteristic morphology and is positive to EDTA, while from zygotene to diplotene the structure of the NOR presents no differences from the rest of the chromatin and only the lateral elements of the synaptonemal complex or its remnants are positive to EDTA. h) The synaptonemal complex runs through the NOR with no differentiation and on occasions its lateral elements show associations with the nucleolus.     

Elevated atmospheric CO2 modifies responses to water-stress and flowering of Mediterranean desert truffle mycorrhizal shrubs

Predicted increases in atmospheric concentration of carbon dioxide (CO₂) coupled with increased temperatures and drought are expected to strongly influence the development of most of the plant species in the world, especially in areas with high risk of desertification like the Mediterranean basin. Helianthemum almeriense is an ecologically important Mediterranean shrub with an added interest because it serves as the host for the Terfezia claveryi mycorrhizal fungus, which is a desert truffle with increasingly commercial interest. Although both plant and fungi are known to be well adapted to dry conditions, it is still uncertain how the increase in atmospheric CO₂ will influence them. In this article we have addressed the physiological responses of H. almeriense × T. claveryi mycorrhizal plants to increases in atmospheric CO₂ coupled with drought and high vapor pressure deficit. This work reports one of the few estimations of mesophyll conductance in a drought deciduous Mediterranean shrub and evaluates its role in photosynthesis limitation. High atmospheric CO₂ concentrations help desert truffle mycorrhizal plants to cope with the adverse effects of progressive drought during Mediterranean springs by improving carbon net assimilation, intrinsic water use efficiency and dispersal of the species through increased flowering events.     

Association between tree-ring and needle δ<Superscript>13</Superscript>C and leaf gas exchange in <Emphasis Type="Italic">Pinus halepensis</Emphasis> under semi-arid conditions

Associations between δ¹³C values and leaf gas exchanges and tree-ring or needle growth, used in ecophysiological compositions, can be complex depending on the relative timing of CO₂ uptake and subsequent redistribution and allocation of carbon to needle and stem components. For palaeoenvironmental and dendroecological studies it is often interpreted in terms of a simple model of δ¹³C fractionation in C₃ plants. However, in spite of potential complicating factors, few studies have actually examined these relationships in mature trees over inter- and intra-annual time-scales. Here, we present results from a 4 years study that investigated the links between variations in leaf gas-exchange properties, growth, and dated δ¹³C values along the needles and across tree rings of Aleppo pine trees growing in a semi-arid region under natural conditions or with supplemental summer irrigation. Sub-sections of tissue across annual rings and along needles, for which time of formation was resolved from growth rate analyses, showed rapid growth and δ¹³C responses to changing environmental conditions. Seasonal cycles of growth and δ¹³C (up to ~4‰) significantly correlated (P<0.01) with photosynthetically active radiation, vapour pressure deficit, air temperature, and soil water content. The irrigation significantly increased leaf net assimilation, stomatal conductance and needle and tree-ring growth rate, and markedly decreased needle and tree-ring δ¹³C values and its sensitivity to environmental parameters. The δ¹³C estimates derived from gas-exchange parameters, and weighted by assimilation, compared closely with seasonal and inter-annual δ¹³C values of needle- and tree-ring tissue. Higher stomatal conductances of the irrigated trees (0.22 vs. 0.08 mol m⁻² s⁻¹ on average) corresponded with ~2.0‰ lower average δ¹³C values, both measured and derived. Derived and measured δ¹³C values also indicated that needle growth, which occurs throughout the stressful summer was supported by carbon from concurrent, low rate assimilation. For Aleppo pine under semi-arid and irrigated conditions, the δ¹³C of tree-ring and needle material proved, in general, to be a reasonable indicator of integrated leaf gas-exchange properties.     

Severe acute respiratory syndrome coronavirus fails to activate cytokine-mediated innate immune responses in cultured human monocyte-derived dendritic cells

Activation of host innate immune responses was studied in severe acute respiratory syndrome coronavirus (SCV)-infected human A549 lung epithelial cells, macrophages, and dendritic cells (DCs). In all cell types, SCV-specific subgenomic mRNAs were seen, whereas no expression of SCV proteins was found. No induction of cytokine genes (alpha interferon [IFN-alpha], IFN-beta, interleukin-28A/B [IL-28A/B], IL-29, tumor necrosis factor alpha, CCL5, or CXCL10) or IFN-alpha/beta-induced MxA gene was seen in SCV-infected A549 cells, macrophages, or DCs. SCV also failed to induce DC maturation (CD86 expression) or enhance major histocompatibility complex class II expression. Our data strongly suggest that SCV fails to activate host cell cytokine gene expression in human macrophages and DCs.