Titanium-45 as a candidate for PET imaging: production, processing & applications

Introduction The 80kD glycoprotein transferrin (TF) and its related receptor (TFR1) play a major role in the recruitment by cancer cells of factors for their multiplication, adhesion, invasion and metastatic potential. Though primarily designed to bind iron and then be internalised into cells with its receptor, TF can also bind most transition metals such as Co, Cr, Mn, Zr, Ni, Cu, V, In & Ga. Under certain conditions TF binds Ti (IV) even more tightly than it does Fe and that this occurs at the N-lobe (as distinct from C) of apoTF. Further, under physiological conditions the species Fe(C)Ti(N)-TF may provide the route for Ti entry into cells via TFR1 (1). Thus, the radiometal PET reporter isotope 45Ti with an ‘intermediate’ (~hrs) half-life suited to tracking cell-focused biological mechanisms is an attractive option for elucidating cellular mechanisms involving TF binding and internalisation, at least in (preclinical) animal models. 45Ti (T½ = 3.08 hr; + branching ratio = 85 %; mean β+ energy = 439keV, no significant dose-conferring non-511keV γ-emissions) was produced using the reaction 45Sc(p,n)45Ti by irradiating (monoisotopic) scandium discs with an energy-degraded proton beam produced by an 18MeV isochronous medical cyclotron. Separation and purification was achieved with an hydroxylamine hydrochloride functionalised resin. Comparative microPET imaging was performed in an immunodeficient mouse model, measuring biodistributions of the radiolabels 45Ti-oxalate and 45Ti-human-TF (45Ti-h-TF), out to 6hr post-injection. Materials and Methods High purity 15mm diameter scandium disc foils (99.5%, Goodfellow, UK) each thickness 0.100 ± 0.005 mm (55 mg) were loaded into an in-house constructed solid-targetry system mounted on a 300mm external beam line utilising helium-gas and chilled water to cool the target body (2). The proton beam was degraded to 11.7 MeV using a graphite disc integrated into the graphite collimator. This energy abolishes the competing ‘contaminant’ reactions 45Sc(p,n+p)44Sc and 45Sc(p,2n)44Ti. Beam current was measured using the well documented 65Cu(p,n)65Zn reaction. Calculations showed that the chosen energy is close to the optimal primary energy (~12 MeV) for maximising the (thin-target) yield from a 0.100 mm thick target. For separation of Ti from the Sc target two methods were examined; (i) ion exchange column separation using 2000 mg AG 50W-X8 resin conditioned with 10mL 9M HCl. Disc is dissolved in 1 mL of 9M HCl, which at completion of reaction is pipetted into column. Successive 1 mL volumes of 9M HCl are added, and subsequent elutions collected. (ii) Following Gagnon et al., (3) a method employing hydroxylamine hydro-chloride functionalised resin (’hydroxamate method’) was applied, similar to its use in our hands for purification and separation of 89Zr (2) following its original description for 89Zr by Holland et al., (4). Disc dissolved in 2mL 6M HCl, then diluted to 2M. Elute through column to waste fraction 1 (w1 – see FIG. 1). Then elute 6 mL of 2M HCl through column to w2, followed by 6 mL of traceSELECT H2O to w3. Finally, elute Ti into successive 1 mL product fractions (p1, 2 etc.) using 5 mL of 1M oxalic acid. This procedure takes approximate 1 hr. 45Ti in elution vials was measured using γ-spectroscopy. Sc in the same vials was determined later using ICP-MS. Results A typical production run using a beam current of 40 μA for 60min on a 0.100mm-thick disc produced an activity of 1.83 GBq. Radionuclidic analysis of an irradiated disc using calibrated cryo-HPGe γ-spectroscopy revealed T½ = 2.97–3.19 hr (95% CI) for 45Ti, and with contaminant 44Sc < 0.19 %, with no other isotopes detected. Despite systematic adjustments to column conditions satisfactory chemical separation was not achieved using the ion exchange column method (i), despite previous reports of its success (5). Typical results of separation using the successful hydroxamate method (ii) are shown on the FIGURE 1. It is seen that significant portion of 45Ti is lost in the initial washing steps leading to waste collection. N = 4 replicate experiments showed a variation (SD) of 10 % of the mean in each elu-tion fraction. Subsequent ICP-MS of the same elutions for (cold) Sc showed approximately 80 % by mass appeared in w1 and 20 % in w2, with negligible total mass (total fraction ~1/6000) of Sc in product (p1–4) vials. However, the FIG. 1 shows that a total of only 30% of the original activity of 45Ti (corrected to EOB) is available in the product vials, with the vial of highest specific activity (p1) containing 14 %. However, using a stack of 2×0.100mm thick Sc discs as a target yields isotope of adequate specific activity with-out need for concentration, for subsequent labelling and small-animal imaging purposes. In a ‘proof-of-principle’ experiment, two groups of healthy Balb/c-nu/nu female adult mice were administered with 45Ti radiotracers. The first group (N = 3) received approximately 20 MBq IP of 45Ti-oxalate buffered to pH = 7.0, and under-went microPET/CT imaging (Super Argus PET, Sedecal, Spain) out to 6hr post-injection, plus biodistribution analysis of radioactivity by dis-section at sacrifice (6hr). The second group (N = 3) received approximately 20 MBq IP of 45Ti-h-TF and were also studied to 6hr post-injection, followed by radioactive analysis after dissection at sacrifice. Organ and tissue biodistributions of the two groups at 6hr were similar but with 45Ti-oxalate showing slightly greater affinity for bone. Biodistribution by dissection results broadly confirmed the findings from PET images. However, TLC results suggested that similarity of radiolabel biodistributions of the two groups may be due to contamination of the TF radiolabel with non-conjugated Ti at time of injection. An alternative explanation is dechelation in vivo of 45Ti from 45Ti-h-TF. Conclusion Despite significant loss of 45Ti to the waste fractions of the separation process (total 53 %, corrected to EOB), 45Ti of acceptable specific activity and high radionuclidic purity has been produced from the reaction 45Sc(p,n)45Ti, with separation and purification of the product by hydroxamate column chemistry, confirming an earlier report. Though microPET in vivo imaging using 45Ti-based radiolabels was shown to be feasible, the similarity in the results for the label 45Ti-h-TF compared with ‘raw’ 45Ti-oxalate suggests further investigations. These may include a direct comparison of in vivo 45Ti-h-TF small-animal imaging plus post-dissection biodistribution with the same procedures using 89Zr labelled h-apotransferrin (6)

Zeros of analytic functions, with or without multiplicities

The classical Mason-Stothers theorem deals with nontrivial polynomial solutions to the equation $a+b=c$. It provides a lower bound on the number of distinct zeros of the polynomial $abc$ in terms of the degrees of $a$, $b$ and $c$. We extend this to general analytic functions living on a reasonable bounded domain $\Omega\subset\mathbb C$, rather than on the whole of $\mathbb C$. The estimates obtained are sharp, for any $\Omega$, and a generalization of the original result on polynomials can be recovered from them by a limiting argument.Comment: This is a retitled and slightly revised version of my paper arXiv:1004.359

Nut production in Bertholletia excelsa across a logged forest mosaic: implications for multiple forest use

Although many examples of multiple-use forest management may be found in tropical smallholder systems, few studies provide empirical support for the integration of selective timber harvesting with non-timber forest product (NTFP) extraction. Brazil nut (Bertholletia excelsa, Lecythidaceae) is one of the world’s most economically-important NTFP species extracted almost entirely from natural forests across the Amazon Basin. An obligate out-crosser, Brazil nut flowers are pollinated by large-bodied bees, a process resulting in a hard round fruit that takes up to 14 months to mature. As many smallholders turn to the financial security provided by timber, Brazil nut fruits are increasingly being harvested in logged forests. We tested the influence of tree and stand-level covariates (distance to nearest cut stump and local logging intensity) on total nut production at the individual tree level in five recently logged Brazil nut concessions covering about 4000 ha of forest in Madre de Dios, Peru. Our field team accompanied Brazil nut harvesters during the traditional harvest period (January-April 2012 and January-April 2013) in order to collect data on fruit production. Three hundred and ninety-nine (approximately 80%) of the 499 trees included in this study were at least 100 m from the nearest cut stump, suggesting that concessionaires avoid logging near adult Brazil nut trees. Yet even for those trees on the edge of logging gaps, distance to nearest cut stump and local logging intensity did not have a statistically significant influence on Brazil nut production at the applied logging intensities (typically 1–2 timber trees removed per ha). In one concession where at least 4 trees ha-1 were removed, however, the logging intensity covariate resulted in a marginally significant (0.09) P value, highlighting a potential risk for a drop in nut production at higher intensities. While we do not suggest that logging activities should be completely avoided in Brazil nut rich forests, when a buffer zone cannot be observed, low logging intensities should be implemented. The sustainability of this integrated management system will ultimately depend on a complex series of socioeconomic and ecological interactions. Yet we submit that our study provides an important initial step in understanding the compatibility of timber harvesting with a high value NTFP, potentially allowing for diversification of forest use strategies in Amazonian Perù

Evaluation of the process of reabilitation of a stretch of Riparian forest in Itapemirim river watershed - ES

Este estudo foi realizado em uma área de floresta ciliar em processo de recuperação mediante reabilitação.A área de estudo está localizada na sub-bacia hidrográfica do rio Itapemirim, no Município de Alegre, ES, Brasil.A ocupação e uso do solo antes da revegetação eram de pastagem com Brachiaria sp. A revegetação da áreafoi feita em 1997, com espécies autóctones e alóctones arbóreas, em arranjo de distribuição aleatório, em umaárea de 1,2 ha. Para a realização dos estudos foram feitos inventários florestais nos períodos de 2004/2005e 2005/2006, sendo medidos os indivíduos de hábito arbustivo e arbóreo com circunferência à altura do peito(CAP) > 5 cm e suas alturas totais. As espécies encontradas na área foram identificadas e classificadas de acordocom seus grupos ecológicos, síndromes de dispersão e presença silvestre, sendo calculados os parâmetros florísticos,a estrutura vertical e a dinâmica estrutural desse povoamento. O objetivo do trabalho foi avaliar o desenvolvimentodo povoamento implantado para subsidiar práticas silviculturais quanto à seleção e implantação de espécies pararevegetação de áreas de floresta ciliar degradadas, em condições semelhantes. Os resultados demonstraram quefoi implantado um povoamento florestal com grande diversidade de espécies e a estratificação em classes dealtura foi à semelhança de povoamentos heterogêneos naturais. As espécies identificadas como edificadoras darevegetação da área estudada foram: Anadenanthera colubrina, Caesalpinia leyostachia, Acacia auriculiformis,Acacia mangium, Handroanthus serratifolius, Inga edulis, Joannesia princeps, Pterogyne nitens, Enterelobiumcontortisiliquum, Tabernaemontana hystrix e Anthocephalus indicus. A distribuição em classes de tamanho dacomunidade implantada ocorre em forma de "J" reverso, havendo a predominância de indivíduos pioneiros emtodas as classes de CAP. A dinâmica da estrutura horizontal apontou que, para o sucesso, continuidade e desenvolvimentoda recuperação da área, seja monitorada a regeneração natural em relação à sua presença e à eficiência dos fatoresbióticos e abióticos que nela interferem. A não observância de indivíduos arbustivos e arbóreos regenerados naturalmente,na classe de inclusão do estudo, indica a fragilidade inicial da área rumo à sustentabilidade do sistema.The study was realized in an area of riparian forest in process by means of rehabilitation. Thestudy area is located in Rio Itapemirim, in Alegre city, Brazil. The occupation and use of the soil beforethe recovery were of pasture with Brachiaria sp. The recovery of this area was in 1997, with autochthonousand allochthonous species, in casually distribution arrangement, in an area of 1,2 ha. For this studies wasrealized forest inventories in the periods of 2004/2005 and 2005/2006, being measured the arboreal individualswith circumference at breast height (CBH) and total heights. The species found in the area were identifiedand classified in this ecological groups, dispersion syndromes and wild presence, it forms calculated thefloristic parameters, the vertical structure and the structural dynamics of this plantation. The objective ofthis work went evaluate the development of the plantation implanted to subsidize practical silviculture withrelationship to the selection and plantation of species for the revegetation of degraded areas of riparianforest, in similar conditions. The results of the studies demonstrated that a forest plantation was implantedwith great diversity of species and the bedding in height classes it went to the likeness of natural heterogeneous.The species identified as builders of the plantation of the studied area were: Anadenanthera colubrina, Caesalpinia leyostachia, Acacia auriculiformis, Acacia mangium, Handroanthus serratifolius, Inga edulis, Joannesia princeps,Pterogyne nitens, Enterelobium contortisiliquum, Tabernaemontana hystrix, and Anthocephalus indicus . Theimplanted community's distribution diametric happens in reverse J shape having the pioneer individuals'predominance in all the classes of CBH. The dynamics of the horizontal structure points that for the success,continuity and development of the recovery of the area, the natural regeneration be monitored in relationto its presence and efficiency of the biotic factors and abiotic those interfere in the same. The not tree individualsobservance in natural regeneration, in the class of inclusion of the study, indicate the initial fragility of thearea heading for sustentabilidade of the system

Structure of Chimpanzee Gut Microbiomes across Tropical Africa

Understanding variation in host-associated microbial communities is important given the relevance of microbiomes to host physiology and health. Using 560 fecal samples collected from wild chimpanzees (Pan troglodytes) across their range, we assessed how geography, genetics, climate, vegetation, and diet relate to gut microbial community structure (prokaryotes, eukaryotic parasites) at multiple spatial scales. We observed a high degree of regional specificity in the microbiome composition, which was associated with host genetics, available plant foods, and potentially with cultural differences in tool use, which affect diet. Genetic differences drove community composition at large scales, while vegetation and potentially tool use drove within-region differences, likely due to their influence on diet. Unlike industrialized human populations in the United States, where regional differences in the gut microbiome are undetectable, chimpanzee gut microbiomes are far more variable across space, suggesting that technological developments have decoupled humans from their local environments, obscuring regional differences that could have been important during human evolution. IMPORTANCE Gut microbial communities are drivers of primate physiology and health, but the factors that influence the gut microbiome in wild primate populations remain largely undetermined. We report data from a continent-wide survey of wild chimpanzee gut microbiota and highlight the effects of genetics, vegetation, and potentially even tool use at different spatial scales on the chimpanzee gut microbiome, including bacteria, archaea, and eukaryotic parasites. Microbial community dissimilarity was strongly correlated with chimpanzee population genetic dissimilarity, and vegetation composition and consumption of algae, honey, nuts, and termites were potentially associated with additional divergence in microbial communities between sampling sites. Our results suggest that host genetics, geography, and climate play a far stronger role in structuring the gut microbiome in chimpanzees than in humans

An empirical evaluation of camera trap study design: How many, how long and when?

Abstract Camera traps deployed in grids or stratified random designs are a well‐established survey tool for wildlife but there has been little evaluation of study design parameters. We used an empirical subsampling approach involving 2,225 camera deployments run at 41 study areas around the world to evaluate three aspects of camera trap study design (number of sites, duration and season of sampling) and their influence on the estimation of three ecological metrics (species richness, occupancy and detection rate) for mammals. We found that 25–35 camera sites were needed for precise estimates of species richness, depending on scale of the study. The precision of species‐level estimates of occupancy (ψ) was highly sensitive to occupancy level, with 0.75) species, but more than 150 camera sites likely needed for rare (ψ < 0.25) species. Species detection rates were more difficult to estimate precisely at the grid level due to spatial heterogeneity, presumably driven by unaccounted habitat variability factors within the study area. Running a camera at a site for 2 weeks was most efficient for detecting new species, but 3–4 weeks were needed for precise estimates of local detection rate, with no gains in precision observed after 1 month. Metrics for all mammal communities were sensitive to seasonality, with 37%–50% of the species at the sites we examined fluctuating significantly in their occupancy or detection rates over the year. This effect was more pronounced in temperate sites, where seasonally sensitive species varied in relative abundance by an average factor of 4–5, and some species were completely absent in one season due to hibernation or migration. We recommend the following guidelines to efficiently obtain precise estimates of species richness, occupancy and detection rates with camera trap arrays: run each camera for 3–5 weeks across 40–60 sites per array. We recommend comparisons of detection rates be model based and include local covariates to help account for small‐scale variation. Furthermore, comparisons across study areas or times must account for seasonality, which could have strong impacts on mammal communities in both tropical and temperate sites