Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

Precise spatial and temporal characterization of cytokinin (CK) responses reveals the function of the CK biosynthesis gene ISOPENTENYLTRANSFERASE 3 during nodule development in Medicago truncatula.     

Clinical versus ultrasound examination in the evaluation of hepatosplenic schistosomiasis mansoni in endemic areas

The best way to appraise the size of abdominal organs remains undefined. Herein we compare the size of liver and spleen in hepatosplenic schistosomiasis using clinical and ultrasound (US) examination, and the size of the organs measured by US with their visualization below the costal margin ("palpable by US"). For this study, 411 individuals from an endemic area for schistosomiasis mansoni in Brazil have been selected. We found that palpable spleens and left liver lobes are larger than non palpable ones. Also, 23% of normal spleens measured by US were palpable on clinical examination, and 22% of spleens increased in size on US were non palpable. A total of 21% of normal spleens were "palpable by US". We also found 54% of normal sized right liver lobes palpable on clinical examination, whilst 54% of the increased livers, measured by US, were non palpable. About 76% of normal right liver lobes were "palpable by US". We conclude that the association of clinical, ultrasound and magnetic resonance imaging (MRI) examinations, in the near future, should give the investigators the necessary tools to perform a more accurate clinical diagnosis of hepatosplenic schistosomiasis mansoni.     

Immunochemical studies on blood groups: The internal structure and immunological properties of water-soluble human blood group A substance studied by Smith degradation,liberation, and fractionation of oligosaccharides and reaction with lectins

Carbohydrate structures in the interior of a blood group A active substance (MSS) were exposed by one and by two Smith degradations. Reactivities of the original glycoprotein and its Smith degraded products with 13 different lectins and with anti-I Ma were studied by quantitative precipitin assay. MSS and its first Smith degraded product completely precipitated Ricinus communis hemagglutinin with five times less of the first Smith degraded glycoprotein being required for 50% precipitation. The second Smith degraded material precipitated only 90% of the lectin. MSS did not precipitate peanut lectin, whereas its first and second Smith degraded products completely precipitated the lectin. The first Smith degraded glycoprotein also reacted well with Wistaria floribunda, Maclura pomifera, Bauhinia purpurea alba, and Geodia lectins indicating that its carbohydrate moiety could contain dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc, dGalβ1 → 3dGlcNAcβ1 → 3dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc determinants at nonreducing ends. The second Smith degraded material precipitated well with Ricinus communis hemagglutinin, Arachis hypogaea, Geodia cydonium, Maclura pomifera, and Helix pomatia lectins showing that dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc residues at terminal nonreducing ends could be involved. Monoclonal anti-I Ma (group 1) serum reacted strongly with the first Smith degraded product indicating large numbers of anti-I Ma determinants, dGalβ1 → 4dGlcNAcβ1 → d 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc at nonreducing ends. The comparable activities of the native and Smith degraded products with wheat germ lectin indicate capacity to react with DGlcNAc residues at nonreducing ends and/or at positions in the interior of the chain. The totality of lectin reactivities indicates heterogeneity of the carbohydrate side chains. Oligosaccharides with ³H at their reducing ends released from the protein core of the first and second Smith degraded products were obtained by treatment with 0.05 m NaOH and 1 M NaB³H₄ at 50 °C for 16 h (Carlson degradation). The liberated reduced oligosaccharides were fractionated by dialysis, followed by retardion, Bio-Gel P-2, P-4, and P-6 columns. They were further purified on charcoal-celite columns, and by preparative paper chromatography and high-pressure liquid chromatography. Their distribution by size was estimated by the yields on dialysis, Bio-Gel P-2, and Bio-Gel P-6 chromatography, and from the radioactivity of the reduced sugars. Of the oligosaccharide fractions from the first Smith degraded product, about 77% of the carbohydrate side chain residues contained from 1 to 6 sugars, 13% from 7 to perhaps 12 sugars, and 10% was nondialyzable (polysaccharides and glycopeptide fragments). Of the second Smith degraded product, approximately 82% of carbohydrate residues had from 1 to 6 sugars, 14% from 7 to perhaps 20 sugars and 4% was nondialyzable. The biological activity profile of the two Smith degraded products together with the size distributions of the oligosaccharides indicated that their carbohydrate side chains, comprised a heterogeneous population ranging in size from 1 to about 12 sugars. When most of these chains that are shorter than hexasaccharides are fully characterized it may be possible to reconstruct the overall structure of the carbohydrate moiety of the blood group substances and account for their biological activities.     

In vitro response to Candida albicans in cultures of whole human blood from young and aged donors

Invasive infections with opportunistic fungi, such as Candida albicans, have become an increasing problem in aged adults in recent years. This work investigates the influence of human ageing on C. albicans recognition by toll-like receptors (TLRs), essential components of the innate immune system, using a cohort of 96 young (15-42 years) and aged (>70 years) human volunteers. No significant differences between aged and young donors were observed on (1) cell surface TLR2, TLR6 and TLR4 expression on lymphocytes, monocytes and granulocytes, (2) production of cytokines [IL-8, IL-1beta, IL-6, IL-10, tumour necrosis factor (TNF)-alpha and IL-12p70] and prostaglandin E(2) (PGE(2)) by whole human blood in response to C. albicans and (3) fungicidal activity of whole blood. A statistically significant higher titre of natural anti-C. albicans antibodies was found in plasma of volunteers between 80 and 95 years old when compared with other age groups, probably as a consequence of the increased levels of serum Ig that has been described in elderly subjects. Therefore, the results indicate that the increased susceptibility to C. albicans infections in the elderly is not a consequence of defects in TLRs expression or signalling, nor of an impaired fungicidal activity of blood.     

Structural insights into dehydratase substrate selection for the borrelidin and fluvirucin polyketide synthases

Engineered polyketide synthases (PKSs) are promising synthetic biology platforms for the production of chemicals with diverse applications. The dehydratase (DH) domain within modular type I PKSs generates an α,β-unsaturated bond in nascent polyketide intermediates through a dehydration reaction. Several crystal structures of DH domains have been solved, providing important structural insights into substrate selection and dehydration. Here, we present two DH domain structures from two chemically diverse PKSs. The first DH domain, isolated from the third module in the borrelidin PKS, is specific towards a trans-cyclopentane-carboxylate-containing polyketide substrate. The second DH domain, isolated from the first module in the fluvirucin B₁ PKS, accepts an amide-containing polyketide intermediate. Sequence-structure analysis of these domains, in addition to previously published DH structures, display many significant similarities and key differences pertaining to substrate selection. The two major differences between BorA DH M3, FluA DH M1 and other DH domains are found in regions of unmodeled residues or residues containing high B-factors. These two regions are located between α3–β11 and β7–α2. From the catalytic Asp located in α3 to a conserved Pro in β11, the residues between them form part of the bottom of the substrate-binding cavity responsible for binding to acyl-ACP intermediates.

     

Parasitism by the mite Acarophenax lacunatus on beetle pests of stored products

Recent reports of the biocontrolpotential of the mite species Acarophenaxlacunatus (Cross & Krantz) (Prostigmata:Acarophenacidae) and the lack of biologicalstudies on this regulatory agent led to thepresent study carried out under laboratoryconditions. The objective of the investigationwas to assess the host range of A. lacunatus,so far only reported as egg parasite ofRhyzopertha dominica (F.) (Coleoptera:Bostrichidae). Four Coleoptera species ofstored cereals were used: R. dominica,Tribolium castaneum (Herbst) (Tenebrionidae),Cryptolestes ferrugineus (Stephens)(Laemophloeidae) and Oryzaephilus surinamensis (L.) (Cucujidae). The highest rates of eggparasitism were observed on R. dominica and T.castaneum, leading to a significant decrease ofpopulations of both species and reduced wheatweight loss. A. lacunatus was also able toparasitize eggs of C. ferrugineus, but not ofO. surinamensis. These results indicate abroader host range of A. lacunatus thaninitially suspected and also strengthen itspossibility of use in integrated pestmanagement programs in storage environments.     

Anti-Sporothrix Antibody Detection in Domestic Cats as an Indicator of a Possible New Occurrence Area for Sporotrichosis in North Brazil

Mycopathologia - Feline sporotrichosis has emerged as an important public health issue in some countries, especially Brazil. Currently, zoonotic transmission of Sporothrix brasiliensis by domestic...     

Contralateral input modulates the excitability of dorsal horn neurons involved in noxious signal processes. Potential role in neuronal sensitization

Wide Dynamic Range (WDR) neurons in the spinal cord receive inputs from the contralateral side that, under normal conditions, are ineffective in generating an active response. These inputs are effective when the target WDRs change their excitability conditions. To further reveal the mechanisms supporting this effectiveness shift, we investigated the weight of the excitation of the contralateral neurons on the target WDR responses. In the circuit of presynaptic (sending) and postsynaptic (receiving) neurons in crossed spinal connections the fibres that form the presynaptic neurons impinge on postsynaptic neurons can be considered the final relay of this contralateral pathway. The enhancement of the presynaptic neuron excitability may thus modify the efficacy of the contralateral input. Pairs of neurons each on a side of the spinal cord, at the L5–L6 lumbar level were simultaneously recorded in intact, anaesthetized, paralysed rats. The excitatory aminoacid NMDA and strychnine, the antagonist of the inhibitory aminoacid glycine, were iontophoretically administrated to presynaptic neurons to increase their excitability. Before and during the drug administration, spontaneous and noxious-evoked activities of the neurons were analysed. During the iontophoresis of the two substances we found that noxious stimuli applied to the receptive field of presynaptic neurons activated up to 50% of the previously unresponsive postsynaptic neurons on the opposite side. Furthermore, the neurons on both sides of the spinal cord showed significantly increased spontaneous activity and amplified responses to ipsilateral noxious stimulation. These findings indicate that the contralateral input participates in the circuit dynamics of spinal nociceptive transmission, by modulating the excitability of the postsynaptic neurons. A possible functional role of such a nociceptive transmission circuit in neuronal sensitization following unilateral nerve injury is hypothesized.     

Assessment of the Relationship between Self-Declared Ethnicity,Mitochondrial Haplogroups and Genomic Ancestry in Brazilian Individuals

In populations that have a high degree of admixture, such as in Brazil, the sole use of ethnicity self-declaration information is not a good method for classifying individuals regarding their ethnicity. Here, we evaluate the relationship of self-declared ethnicities with genomic ancestry and mitochondrial haplogroups in 492 individuals from southeastern Brazil. Mitochondrial haplogroups were obtained by analyzing the hypervariable regions of the mitochondrial DNA (mtDNA), and the genomic ancestry was obtained using 48 autosomal insertion-deletion ancestry informative markers (AIM). Of the 492 individuals, 74.6% self-declared as White, 13.8% as Brown and 10.4% as Black. Classification of the mtDNA haplogroups showed that 46.3% had African mtDNA, and the genomic ancestry analysis showed that the main contribution was European (57.4%). When we looked at the distribution of mtDNA and genomic ancestry according to the self-declared ethnicities from 367 individuals who self-declared as White, 37.6% showed African mtDNA, and they had a high contribution of European genomic ancestry (63.3%) but also a significant contribution of African ancestry (22.2%). Of the 68 individuals who self-declared as Brown, 25% showed Amerindian mtDNA and similar contribution of European and African genomic ancestries. Of the 51 subjects who self-declared as black, 80.4% had African mtDNA, and the main contribution of genomic ancestry was African (55.6%), but they also had a significant proportion of European ancestry (32.1%). The Brazilian population had a uniform degree of Amerindian genomic ancestry, and it was only with the use of genetic markers (autosomal or mitochondrial) that we were able to capture Amerindian ancestry information. Additionally, it was possible to observe a high degree of heterogeneity in the ancestry for both types of genetic markers, which shows the high genetic admixture that is present in the Brazilian population. We suggest that in epidemiological studies, the use of these methods could provide complementary information.