Novel DOTA-neurotensin analogues for 111In scintigraphy and 68Ga PET imaging of neurotensin receptor-positive tumors

Overexpression of the high affinity neurotensin receptor 1 (NTSR1), demonstrated in several human cancers, has been proposed as a new marker for human ductal pancreatic carcinoma and as an independent factor for poor prognosis for ductal breast cancer, head and neck squamous cell carcinoma, and non-small cell lung cancer. The aim of the present study was to develop new DOTA-neurotensin analogues for positron emission tomography (PET) imaging with (68)Ga and for targeted radiotherapy with (90)Y or (177)Lu. We synthesized a DOTA-neurotensin analogue series. Two of these peptides bear two sequence modifications for metabolic stability: DOTA-NT-20.3 shares the same peptide sequence as the previously described DTPA-NT-20.3. In the sequence of DOTA-NT-20.4, the Arg(8)-Arg(9) bond was N-methylated instead of the Pro(7)-Arg(8) bond in DOTA-NT-20.3. An additional sequence modification was introduced in DOTA-LB119 to increase stability. A spacer was added between DOTA and the peptide sequence to increase affinity. Binding to HT29 cells, which express NTSR1, in vivo stability, and biodistribution of the various analogues were compared, and the best candidate was used to image tumors of various sizes with the microPET in mice. (111)In-DOTA-NT-20.3, in spite of a relatively high uptake in kidneys, showed specific tumor uptake and elevated tumor to other organ uptake ratios. High contrast images were obtained at early time points after injection that allowed tumor detection at a time interval postinjection appropriate for imaging with the short-lived radionuclide (68)Ga. (111)In-DOTA-NT-20.4 displayed inferior binding to HT29 cells and reduced tumor uptake. (111)In-DOTA-LB119 displayed at early time points a significantly lower renal uptake but also a lower tumor uptake than (111)In-DOTA-NT-20.3, although binding to HT29 cells was similar. (68)Ga-DOTA-NT-20.3 displayed higher tumor uptake than (68)Ga-DOTA-LB119 and allowed the detection of very small tumors by PET. In conclusion, DOTA-NT-20.3 is a promising candidate for (68)Ga-PET imaging of neurotensin receptor-positive tumors. DOTA-NT-20.3 may also be considered for therapy, as the yttrium-labeled peptide has higher affinity than that of the indium-labeled one. A prerequisite for therapeutic application of this neurotensin analogue would be to lower kidney uptake, for example, by infusion of basic amino acids, gelofusin, or albumin fragments, to prevent nephrotoxicity, as with radiolabeled somatostatin analogues.     

GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the β-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of ‘Hamlin’, ‘Pera’, and ‘Valencia’ sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08?% among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.     

Improving Serodiagnosis of Human and Canine Leishmaniasis with Recombinant Leishmania braziliensis Cathepsin L-like Protein and a Synthetic Peptide Containing Its Linear B-cell Epitope

Background

The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis.

Methodology/Principal Findings

We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis.

Conclusions/Significance

The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.     

Spider communities in two plant architectures of epiphytic cacti in the Brazilian Atlantic forest

The vegetative architecture of the Cactaceae provides sites used by spiders as hideout, foraging, mating and oviposition. We found more web weavers in plants with cylindrical cladodia and more hunters in plants with flat cladodia. Between February 2014 and January 2015 1551 spiders distributed in 20 families were collected: one of these families, Palpimanidae, had only juvenile individuals collected; of the other 19 families, 80 species/morphospecies were identified. This work aims to contribute to the knowledge of biodiversity in the canopy of the Atlantic Forest, especially the relationship between cacti and spiders.     

Analysis of glucose metabolism in farmed European sea bass (Dicentrarchus labrax L.) using deuterated water

Glucose metabolism in free-swimming fasted and fed seabass was studied using deuterated water ((2)H(2)O). After transfer to seawater enriched with 4.9% (2)H(2)O for 6-h or for 72-h, positional and mole percent enrichment (MPE) of plasma glucose and water were quantified by (2)H NMR and ESI-MS/MS. Plasma water (2)H-enrichment reached that of seawater within 6h. In both fasted and fed fish, plasma glucose MPE increased asymptotically attaining ~55% of plasma water enrichment by 72 h. The distribution of (2)H-enrichment between the different glucose positions was relatively uniform. The gluconeogenic contribution to glucose that was synthesized during (2)H(2)O administration was estimated from the ratio of position 5 and 2 glucose enrichments. For both fed and fasted fish, gluconeogenesis accounted for 98±1% of the glucose that was produced during the 72-h (2)H(2)O administration period. For fasted fish, gluconeogenic contributions measured after 6h were identical to 72-h values (94±3%). For fed fish, the apparent gluconeogenic contribution at 6-h was significantly lower compared to 72-h (79±5% versus 98±1%, p<0.05). This may reflect a brief augmentation of gluconeogenic flux by glycogenolysis after feeding and/or selective enrichment of plasma glucose position 2 via futile glucose-glucose-6-phosphate cycling.     

A study on the environmental aspects of WEEE plastic recycling in a Brazilian company

Purpose

The high consumption of electrical and electronic equipment motivated by the rapid technological advances seen over the years has lead to an increase in the generation of waste electrical and electronic equipment (WEEE). Such residues contain various dangerous substances and therefore deserve special attention. To that end, the Brazilian Policy on Solid Waste has provided guidelines on integrated and solid waste management, such as consumer electronics, aiming at their appropriate disposal and treatment through reverse logistics. In this context, the present work focuses on studying the recycling of some WEEE plastics.

Methods

This study was conducted using the methodological framework presented in the International Standard ISO 14040:2006 and aimed to determine the life cycle inventory (LCI) of a WEEE plastic recycling process in a company in Brazil. Having collected the data, it was possible to identify and quantify the environmental aspects caused by the recycling process of major plastics (acrylonitrile-butadiene-styrene (ABS) and high impact polystyrene (HIPS). The study was conducted in the only company in Brazil that operates WEEE plastic recycling in large scale.

Results and discussion

Some of the environmental aspects caused during the recycling process of the plastics under study were identified and quantified. As a result, besides presenting the inventory, it was also possible to determine a reduction in the consumption of energy and in CO₂ emissions. When compared to the production of virgin ABS and HIPS, the recycling processes for such plastics showed a reduction in energy consumption by approximately 90% for both plastics and a reduction in CO₂ emissions by approximately 84% for HIPS and 87% for ABS. The plastics recycled by the company retain over 90% of their virgin mechanical properties.

Conclusions

The study shows that recycling is highly relevant and that components present in WEEE received appropriate destination and treatment. Recycling avoids environmental impacts as it prevents WEEE from being disposed of in landfills and as the pellets of recycled plastics can re-enter the supply chain as raw materials. Considering the legislation in Brazil, the stage of collection/transport/treatment of WEEE conducted by the company under study presents strong indications of contributions to the environment, society, and economy of the country.

     

South Brazilian wines: culturable yeasts associated to bottled wines produced in Rio Grande do Sul and Santa Catarina

A comprehensive understanding of the presence and role of yeasts in bottled wines helps to know and control the organoleptic quality of the final product. The South Region of Brazil is an important wine producer, and the state of “Rio Grande do Sul” (RS) accounts for 90% of Brazilian wines. The state of “Santa Catarina” (SC) started the production in 1975, and is currently the fifth Brazilian producer. As there is little information about yeasts present in Brazilian wines, our main objective was to assess the composition of culturable yeasts associated to bottled wines produced in RS and SC, South of Brazil. We sampled 20 RS and 29 SC bottled wines produced between 2003 and 2011, and we isolated culturable yeasts in non-selective agar plates. We identified all isolates by sequencing of the D1/D2 domain of LSU rDNA or ITS1-5.8 S-ITS2 region, and comparison with type strain sequences deposited in GenBank database. Six yeast species were shared in the final product in both regions. We obtained two spoilage yeast profiles: RS with Zygosaccharomyces bailii and Pichia membranifaciens (Dekkera bruxellensis was found only in specific table wines); and SC with Dekkera bruxellensis and Pichia manshurica. Knowledge concerning the different spoilage profiles is important for winemaking practices in both regions.     

Factorial design and response surface optimization of crude violacein for Chromobacterium violaceum production

Initially an eleven variable, sixteen assay 2^15–11 fractional factorial design, was used to determine the key factors in the production of violacein produced by Chromobacterium violaceum, CCT 3496. Subsequently five and three factor central composite designs were executed to determine response surfaces with the aim of optimizing cellular mass and crude violacein production. The 7.5 g l^–1 dry cell mass and 0.17 g l^–1 crude violacein productions obtained with our initial culture medium were increased to 21 g l^–1 and 0.43 g l^–1, respectively, for a medium investigated in the three factor design.     

Are Brazilian cervids at risk of prion diseases?

Prion diseases are neurodegenerative fatal disorders that affect human and non-human mammals. Chronic Wasting Disease (CWD) is a prion disease of cervids regarded as a public health problem in North America, and polymorphisms at specific codons in the PRNP gene are associated with this disease. To assess the potential CWD susceptibility of South American free-ranging deer, the presence of these polymorphisms was examined in Mazama gouazoubira, Ozotoceros bezoarticus and Blastocerus dichotomus. Despite the lack of CWD reports in Brazil, the examined codons (95, 96, 116, 132, 225, and 226) of the PRNP gene showed potential CWD susceptibility in Brazilian deer. Low abundancy of deer in Brazil possibly difficult both CWD proliferation and detection, however, CWD surveillance may not be neglected.