Heterologous Assay Research Articles

Evaluation of functionality of type II toxin-antitoxin systems of Clostridioides difficile R20291

Clostridioides difficile is a nosocomial, Gram-positive, strictly anaerobic, spore-forming pathogen capable of colonizing and proliferating in the human intestine. In bacteria, it has been shown that the Toxin-Antitoxin systems mediate the cellular response to external stress by initiating processes such as biofilm formation and programmed cell death. This work aims to evaluate the functionality of four type II TA modules of Clostridioides difficile R20291. We performed bioinformatic analysis to search for putative TA systems using the TADB platform. Then we performed a heterologous expression assay to evaluate the functionality of these systems. Our results showed that the MazEF and RelBE systems were functional, suggesting that their corresponding toxins possess an endoribonuclease activity. In conclusion, MazEF and RelBE systems of C. difficile R20291 are functional in a heterologous expression system.

Widespread Inhibition, Antagonism, and Synergy in Mouse Olfactory Sensory Neurons In Vivo

Sensory information is selectively or non-selectively enhanced and inhibited in the brain, but it remains unclear whether and how this occurs at the most peripheral level. Using invivo calcium imaging of mouse olfactory bulb and olfactory epithelium in wild-type and mutant animals, we show that odors produce not only excitatory but also inhibitory responses in olfactory sensory neurons (OSNs). Heterologous assays indicate that odorants can act as agonists to some but inverse agonists to other odorant receptors. We also demonstrate that responses to odor mixtures are extensively suppressed or enhanced in OSNs. When high concentrations of odors are mixed, widespread antagonism suppresses the overall response amplitudes and density. In contrast, a mixture of low concentrations of odors often produces synergistic effects and boosts the faint odor inputs. Thus, odor responses are extensively tuned by inhibition, antagonism, and synergy at the most peripheral level, contributing to robust sensory representations.

A cell suspension based uptake method to study high affinity glucosinolate transporters

BackgroundGlucosinolates are an important class of secondary metabolites characteristic to the order Brassicales. They are known to play a major role in plant defense and from the human perspective, can be anticarcinogenic or antinutritive. GTRs are plasma-membrane localized high affinity glucosinolate transporters, which are important components of the source (leaf) to sink (seed) translocation of intact glucosinolates in members of Brassicaceae family. GTRs are identified as major candidates for Brassica crop improvement, thus dictating a need for their functional characterization. However, currently there are limitations in availability of heterologous assay systems for functional characterization of plant secondary metabolite transporters. To date, the animal-based Xenopus oocyte system is the best established heterologous system for functional characterization of these transporters. Inherent biochemical and physiological attributes unique to the plant membranes necessitate the need for developing plant-based transporters assay systems as well.MethodsIn this study, Agrobacterium mediated transformation was used to develop GTR expressing cotton cell lines (CCL-1) for functional characterization of the Arabidopsis high affinity glucosinolate transporters, AtGTR1 and AtGTR2. Following sub-cellular localization of AtGTRs, we standardized the glucosinolate uptake assays using cell suspension cultures of AtGTR expressing CCL-1 its requirement of pH, salt, and time based glucosinolate uptake. Using the GTR expressing CCL-1, we subsequently performed kinetic analysis of AtGTR1 and AtGTR2 for different glucosinolate substrates, sinigrin, gluconapin and sinalbin.ResultsSeveral clones expressing each of AtGTR1 and AtGTR2 were obtained showing high level of GTR expression and were maintained through regular sub-culturing. Both AtGTR1 and AtGTR2 are predominantly plasma-localized proteins when overexpressed in CCL-1 cells. Uptake assays were standardized, suggesting that glucosinolate uptake of GTR expressing CCL-1 is robust within the physiological pH range 5–6, and at lower concentration of nitrate salts. GTR expressing CCL-1 cells show increasing glucosinolate accumulation in time course experiment. Kinetic studies over a wide glucosinolate concentrations (10–800 µM) revealed that our novel assay system displayed robust GTR-mediated uptake of different glucosinolates and unambiguously helps elucidate the saturable kinetics of GTRs. Our system confirms the high affinity of AtGTRs for both aliphatic and aromatic glucosinolates.ConclusionThe transporter assay system described in this study holds potential for studying sub-functionalization amongst GTR homologs present across Brassicaceae family. The fast growing CCL-1 cells, confer the benefits of an in vitro system for quick assays and is plant based thus enabling optimal expression without sequence modifications. The efficient functioning of the GTR transporters in the heterologous CCL-1 opens the possibility of using this plant cell suspension system for functional characterization of other metabolite transporters.

Functional characterisation of fatty acyl desaturase, Fads2, and elongase, Elovl5, in the Boddart's goggle-eyed goby Boleophthalmus boddarti (Gobiidae) suggests an incapacity for long-chain polyunsaturated fatty acid biosynthesis.

The biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA), a process to convert C18 polyunsaturated fatty acids into eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or arachidonic acid (ARA), requires the concerted activities of two enzymes, the fatty acyl desaturase (Fads) and elongase (Elovl). This study highlights the cloning, functional characterisation and tissue expression pattern of a Fads and an Elovl from the Boddart's goggle-eyed goby (Boleophthalmus boddarti), a mudskipper species widely distributed in the Indo-Pacific region. Phylogenetic analysis revealed that the cloned fads and elovl are clustered with other teleost orthologs, respectively. The investigation of the genome of several mudskipper species, namely Boleophthalmus pectinirostris, Periophthalmus schlosseri and Periophthalmus magnuspinnatus, revealed a single Fads2 and two elongases, Elovl5 and Elovl4 for each respective species. A heterologous yeast assay indicated that the B. boddarti Fads2 possessed low desaturation activity on C18 PUFA and no desaturation on C20 and C22 PUFA substrates. In comparison, the Elovl5 showed a wide range of substrate specificity, with a capacity to elongate C18, C20 and C22 PUFA substrates. An amino acid residue that affects the capacity to elongate C22:5n-3 was identified in the B. boddarti Elovl5. Both genes are highly expressed in brain tissue. Among all tissues, DHA is highly concentrated in neuron-rich tissues, whereas EPA is highly deposited in gills. Taken together, the results showed that due to the inability to perform desaturation steps, B. boddarti is unable to biosynthesise LC-PUFA, relying on dietary intake to acquire these nutrients.

Functional characterisation and in silico modelling of MdPSY2 variants and MdPSY5 phytoene synthases from Malus domestica

Carotenoids are plastid isoprenoid pigments that play critical roles in light harvesting, photoprotection, and phytohormone biosynthesis. They are also vitamin-A precursors and antioxidant molecules important for human nutrition. Apples (e.g. Malus x domestica Borkh), one of the most widely consumed fruits with high nutrient levels, have a very low carotenoid concentration in flesh, compared with other fruits and vegetables. This could be explained by a deficiency in carotenoid synthesis/accumulation and/or accelerated degradation. We analysed the contribution of M. domestica cv. ‘Fuji’ phytoene synthase (PSY) in the biosynthesis of carotenoids and determined that among four MdPSY genes present in the organism, MdPSY2 and MdPSY5 are highly expressed in leaves and during fruit ripening in line with an increment in carotenoid content in fruits. Furthermore, two representative polymorphic MdPSY2 variants were found, one with a Tyr358Phe substitution (MdPSY2_F) and the other that additionally has a six-amino-acid deletion in the signal peptide (MdPSY2_CG). MdPSY2, MdPSY5, MdPSY2_F and MdPSY2_CG are all localised in plastids. Interestingly, the polymorphic MdPSY2_F and MdPSY2_CG variants show lower enzymatic activity than the wild-type form in a heterologous complementation assay, which could be attributed to the Tyr358Phe substitution close to the active-site pocket, as was suggested by 3-D modelling analysis. The presence of polymorphic MdPSY2 variants with lower enzymatic activity could be partially responsible for the low carotenoid content in Fuji apple fruits.

Fluorescent analyses of Bacillus thuringiensis Cry1Fa and Cry1Ab toxin binding sites on brush border membrane vesicles of Ostrinia nubilalis (Hübner), Diatraea grandiosella (Dyar), and Helicoverpa zea (Boddie) larvae

Bacillus thuringiensis (Bt) Cry1Fa and Cry1Ab proteins are important Cry toxins due to their high, selective toxicity against a number of lepidopteran species, including important pests of corn and cotton. Competition binding assays are a classical tool for investigating Cry toxin interactions with target pest insects. We developed a fluorescence-based binding assay and assessed Cry1Fa and Cry1Ab toxin binding to brush border membrane preparations from lepidopteran corn pests including Ostrinia nubilalis (European corn borer, ECB), Diatraea grandiosella (south western corn borer, SWCB), and Helicoverpa zea (corn earworm, CEW). Homologous and heterologous competition binding assays with fluorophore-(Alexa488)-labeled Cry1Fa toxin showed that Cry1Fa shares binding site(s) with Cry1Ab toxin in ECB, and SWCB for which Cry1Ab has higher affinity than Cry1Fa. Apart from the shared binding sites, Cry1Ab and Cry1Fa bind an additional site(s) in ECB and SWCB. In CEW, Cry1Fa and Cry1Ab each, has a high affinity binding site(s), which binds the heterologous toxin with low affinity. The Cry1Ab-Cry1Fa toxin binding models for ECB, SWCB and CEW based on our results are considered in the context of what is known about acquired cross-resistance against Cry1Ab and Cry1Fa toxins.

Transcriptome profiling of Fagopyrum tataricum leaves in response to lead stress

BackgroundLead (Pb) pollution is a widespread environmental problem that is harmful to living organisms. Tartary buckwheat (Fagopyrum tataricum), a member of the family Polygonaceae, exhibits short growth cycles and abundant biomass production, could be an ideal plant for phytoremediation due to its high Pb tolerance. Here, we aimed to explore the molecular basis underlying the responses of this plant to Pb stress.ResultsIn our study, ultrastructural localization assays revealed that Pb ions primarily accumulate in leaf vacuoles. RNA deep sequencing (RNA-Seq) of tartary buckwheat leaves was performed on two Pb-treated samples, named Pb1 (2000 mg/kg Pb (NO3)2) and Pb2 (10,000 mg/kg Pb (NO3)2), and a control (CK). A total of 88,977 assembled unigenes with 125,203,555 bases were obtained. In total, 2400 up-regulated and 3413 down-regulated differentially expressed genes (DEGs) were identified between CK and Pb1, and 2948 up-regulated DEGs and 3834 down-regulated DEGs were generated between CK and Pb2, respectively. Gene Ontology (GO) and pathway enrichment analyses showed that these DEGs were primarily associated with ‘cell wall’, ‘binding’, ‘transport’, and ‘lipid and energy’ metabolism. The results of quantitative real-time PCR (qRT-PCR) analyses of 15 randomly selected candidate DEGs and 6 regulated genes were consistent with the results of the transcriptome analysis. Heterologous expression assays in the yeast strain Δycf1 indicated that overexpressing CCCH-type zinc finger protein 14 (ZFP14) enhanced sensitivity to Pb2+, while 5 other genes, namely, metal transporter protein C2 (MTPC2), phytochelatin synthetase-like family protein (PCSL), vacuolar cation/proton exchanger 1a (VCE1a), natural resistance-associated macrophage protein 3 (Nramp3), and phytochelatin synthetase (PCS), enhanced the Pb tolerance of the mutant strain.ConclusionCombining our findings with those of previous studies, we generated a schematic model that shows the metabolic processes of tartary buckwheat under Pb stress. This study provides important data for further genomic analyses of the biological and molecular mechanisms of Pb tolerance and accumulation in tartary buckwheat.

The Integrity of the Cell Wall and Its Remodeling during Heterocyst Differentiation Are Regulated by Phylogenetically Conserved Small RNA Yfr1 in Nostoc sp. Strain PCC 7120.

Yfr1 is a strictly conserved small RNA in cyanobacteria. A bioinformatic prediction to identify possible interactions of Yfr1 with mRNAs was carried out by using the sequences of Yfr1 from several heterocyst-forming strains, including Nostoc sp. strain PCC 7120. The results of the prediction were enriched in genes encoding outer membrane proteins and enzymes related to peptidoglycan biosynthesis and turnover. Heterologous expression assays with Escherichia coli demonstrated direct interactions of Yfr1 with mRNAs of 11 of the candidate genes. The expression of 10 of them (alr2458, alr4550, murC, all4829, all2158, mraY, alr2269, alr0834, conR, patN) was repressed by interaction with Yfr1, whereas the expression of amiC2, encoding an amidase, was increased. The interactions between Yfr1 and the 11 mRNAs were confirmed by site-directed mutagenesis of Yfr1. Furthermore, a Nostoc strain with reduced levels of Yfr1 had larger amounts of mraY and murC mRNAs, supporting a role for Yfr1 in the regulation of those genes. Nostoc strains with either reduced or increased expression of Yfr1 showed anomalies in cell wall completion and were more sensitive to vancomycin than the wild-type strain. Furthermore, growth in the absence of combined nitrogen, which involves the differentiation of heterocysts, was compromised in the strain overexpressing Yfr1, and filaments were broken at the connections between vegetative cells and heterocysts. These results indicate that Yfr1 is an important regulator of cell wall homeostasis and correct cell wall remodeling during heterocyst differentiation.IMPORTANCE Bacterial small RNAs (sRNAs) are important players affecting the regulation of essentially every aspect of bacterial physiology. The cell wall is a highly dynamic structure that protects bacteria from their fluctuating environment. Cell envelope remodeling is particularly critical for bacteria that undergo differentiation processes, such as spore formation or differentiation of heterocysts. Heterocyst development involves the deposition of additional layers of glycolipids and polysaccharides outside the outer membrane. Here, we show that a cyanobacterial phylogenetically conserved small regulatory RNA, Yfr1, coordinates the expression of proteins involved in cell wall-related processes, including peptidoglycan metabolism and transport of different molecules, as well as expression of several proteins involved in heterocyst differentiation.

The Pkn22 Kinase of Nostoc PCC 7120 Is Required for Cell Differentiation via the Phosphorylation of HetR on a Residue Highly Conserved in Genomes of Heterocyst-Forming Cyanobacteria

Hanks-type kinases encoding genes are present in most cyanobacterial genomes. Despite their widespread pattern of conservation, little is known so far about their role because their substrates and the conditions triggering their activation are poorly known. Here we report that under diazotrophic conditions, normal heterocyst differentiation and growth of the filamentous cyanobacterium Nostoc PCC 7120 require the presence of the Pkn22 kinase, which is induced under combined nitrogen starvation conditions. By analyzing the phenotype of pkn22 mutant overexpressing genes belonging to the regulatory cascade initiating the development program, an epistatic relationship was found to exist between this kinase and the master regulator of differentiation, HetR. The results obtained using a bacterial two hybrid approach indicated that Pkn22 and HetR interact, and the use of a genetic screen inducing the loss of this interaction showed that residues of HetR which are essential for this interaction to occur are also crucial to HetR activity both in vitro and in vivo. Mass spectrometry showed that HetR co-produced with the Pkn22 kinase in Escherichia coli is phosphorylated on Serine 130 residue. Phosphoablative substitution of this residue impaired the ability of the strain to undergo cell differentiation, while its phosphomimetic substitution increased the number of heterocysts formed. The Serine 130 residue is part of a highly conserved sequence in filamentous cyanobacterial strains differentiating heterocysts. Heterologous complementation assays showed that the presence of this domain is necessary for heterocyst induction. We propose that the phosphorylation of HetR might have been acquired to control heterocyst differentiation.

Functional Multigenomic Screening of Human-Associated Bacteria for NF-κB-Inducing Bioactive Effectors.

The effect of the microbiota on its human host is driven, at least in part, by small-molecule and protein effectors it produces. Here, we report on the use of functional multigenomic screening to identify microbiota-encoded effectors. In this study, genomic DNA from 116 human-associated bacteria was cloned en masse, and the resulting multigenomic library was screened using a nuclear factor-κB reporter (NF-κB) assay. Functional multigenomics builds on the concept of functional metagenomics but takes advantage of increasing advances in cultivating and sequencing human-associated bacteria. Effector genes found to confer NF-κB-inducing activity to Escherichia coli encode proteins in four general categories: cell wall hydrolases, membrane transporters, lipopolysaccharide biosynthetic enzymes, and proteins of unknown function. The compact nature of multigenomic libraries, which results from the ability to normalize input DNA ratios, should simplify screening of libraries using diverse heterologous hosts and reporter assays, increasing the rate of discovery of novel effector genes.IMPORTANCE Human-associated bacteria are thought to encode bioactive small molecules and proteins that play an intimate role in human health and disease. Here, we report on the creation and functional screening of a multigenomic library constructed using genomic DNA from 116 bacteria found at diverse sites across the human body. Individual clones were screened for genes capable of conferring NF-κB-inducing activity to Escherichia coli NF-κB is a useful reporter for a range of cellular processes related to immunity, pathogenesis, and inflammation. Compared to the screening of metagenomic libraries, the ability to normalize input DNA ratios when constructing a multigenomic library should facilitate the more efficient examination of commensal bacteria for diverse bioactivities. Multigenomic screening takes advantage of the growing available resources in culturing and sequencing the human microbiota and generates starting points for more in-depth studies on the mechanisms by which commensal bacteria interact with their human host.

The grapevine R2R3-type MYB transcription factor VdMYB1 positively regulates defense responses by activating the stilbene synthase gene 2 (VdSTS2)

BackgroundResveratrol is a naturally occurring plant stilbene that exhibits a wide range of valuable biological and pharmacological properties. Although the beneficial effects of trans-resveratrol to human health and plant protection against fungal pathogens and abiotic stresses are well-established, yet little is known about the molecular mechanisms regulating stilbene biosynthesis in plant defense progress.ResultsHere, we cloned and identified the Chinese wild grape (Vitis davidii) R2R3-MYB transcription factor VdMYB1, which activates defense responses against invading pathogen. VdMYB1 transcripts were significantly upregulated after inoculation with the grapevine powdery mildew fungus Erysiphe necator (Schw.) Burr. Transient expression analysis using onion epidermal cells and Arabidopsis thaliana protoplasts showed that VdMYB1 was localized in the nucleus. Yeast one-hybrid assays revealed that VdMYB1 acts as a transcriptional activator. Grapevine leaves transiently overexpressing VdMYB1 showed a lower number of fungal conidiophores compared with wild-type leaves. Overexpression of VdMYB1 in grapevine leaves did not alter the expression of genes in salicylic acid- and jasmonate-dependent pathways, but affected the expression of stilbene synthase (STS) genes, key regulators of flavonoid metabolism. Results of electrophoretic mobility shift assays and in vivo transcriptional activation assays showed that VdMYB1 binds to the MYB binding site (MYBBS) in the STS2 gene promoter, thus activating STS2 transcription. In heterologous expression assays using tobacco leaves, VdMYB1 activated STS2 gene expression and increased the accumulation of resveratrol.ConclusionsOur study showed that VdMYB1 activates STS2 gene expression to positively regulate defense responses, and increases the content of resveratrol in leaves.

Insight Into Mosquito GnRH-Related Neuropeptide Receptor Specificity Revealed Through Analysis of Naturally Occurring and Synthetic Analogs of This Neuropeptide Family.

Adipokinetic hormone (AKH), corazonin (CRZ), and the AKH/CRZ-related peptide (ACP) are neuropeptides considered homologous to the vertebrate gonadotropin-releasing hormone (GnRH). All three Aedes aegypti GnRH-related neuropeptide receptors have been characterized and functionally deorphanized. Individually they exhibit high specificity for their native ligands, prompting us to investigate the contribution of ligand structures in conferring receptor specificity for two of these receptors. Here, we designed a series of analogs based on the native ACP sequence and screened them using a heterologous system to identify critical residues required for ACP receptor (ACPR) activation. Analogs lacking the carboxy-terminal amidation, replacing aromatics, as well as truncated analogs were either completely inactive or had very low activities on ACPR. The polar threonine (position 3) and the blocked amino-terminal pyroglutamate are also critical, whereas ACP analogs with alanine substitutions at position 2 (valine), 5 (serine), 6 (arginine), and 7 (aspartate) were less detrimental including the substitution of charged residues. Replacing asparagine (position 9) with an alanine resulted in a 5-fold more active analog. A naturally-occurring ACP analog, with a conserved substitution in position two, was well tolerated yet displayed significantly reduced activity compared to the native mosquito ACP peptide. Chain length contributes to ligand selectivity in this system, since the endogenous octapeptide Aedae-AKH does not activate the ACPR whereas AKH decapeptides show low albeit significant activity. Similarly, we utilized this in vitro heterologous assay approach against an A. aegypti AKH receptor (AKHR-IA) testing carefully selected naturally-occurring AKH analogs from other insects to determine how substitutions of specific residues in the AKH ligand influence AKHR-IA activation. AKH analogs having single substitutions compared to Aedae-AKH revealed position 7 (either serine or asparagine) was well tolerated or had slightly improved activation whereas changes to position 6 (proline) compromised receptor activation by nearly 10-fold. Substitution of position 3 (threonine) or analogs with combinations of substitutions were quite detrimental with a significant decrease in AKHR-IA activation. Collectively, these results advance our understanding of how two GnRH-related systems in A. aegypti sharing the most recent evolutionary origin sustain independence of function and signaling despite their relatively high degree of ligand and receptor homology.

Deep-intronic variants in CNGB3 cause achromatopsia by pseudoexon activation.

Our comprehensive cohort of 1100 unrelated achromatopsia (ACHM) patients comprises a considerable number of cases (~5%) harboring only a single pathogenic variant in the major ACHM gene CNGB3. We sequenced the entire CNGB3 locus in 33 of these patients to find a second variant which eventually explained the patients' phenotype. Forty-seven intronic CNGB3 variants were identified in 28 subjects after a filtering step based on frequency and the exclusion of variants found in cis with pathogenic alleles. In a second step, in silico prediction tools were used to filter out those variants with little odds of being deleterious. This left three variants that were analyzed using heterologous splicing assays. Variant c.1663-1205G>A, found in 14 subjects, and variant c.1663-2137C>T, found in two subjects, were indeed shown to exert a splicing defect by causing pseudoexon insertion into the transcript. Subsequent screening of further unsolved CNGB3 subjects identified four additional cases harboring the c.1663-1205G>A variant which makes it the eighth most frequent CNGB3 variant in our cohort. Compound heterozygosity could be validated in ten cases. Our study demonstrates that whole gene sequencing can be a powerful approach to identify the second pathogenic allele in patients apparently harboring only one disease-causing variant.

A Novel Effector Protein of Apple Proliferation Phytoplasma Disrupts Cell Integrity of Nicotiana spp. Protoplasts.

Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by ‘Candidatus Phytoplasma mali’ (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called “Protein in Malus Expressed 2” (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.

Cloning and Functional Assessments of Floral-Expressed SWEET Transporter Genes from Jasminum sambac.

Sugar transporters of the SWEET family mediate cross membrane movement of mono- and disaccharides and play vital roles in diverse physiological and pathophysiological processes, including sink–source relationship, pathogen responses, reproductive growth, and development. However, it remains to be determined how these transporters function in non-module plants of agricultural significance, given the evolutionarily diverse traits. In this study, we combined transcriptome analysis, rapid amplification of cDNA ends-cloning (RACE-cloning), expression profiling, and heterologous functional assay to identify SWEET genes that may have potential roles during flower opening and sexual reproduction in Jasminum sambac . During the anthesis, the floral organs of J. sambac express seven SWEET homologous genes from all four clades of the family. JsSWEET9 and 2 are significantly upregulated when flowers are fully opened, up to 6- and 3-fold compared to unopened buds, respectively. The other transporters, JsSWEET1, 5, 10, and 17 are also accumulated slightly at stage associated with fragrance release, whereas only the vacuole transporter JsSWEET16 showed small decrease in transcript level after anthesis. The JsSWEET5, a clade II member, is capable to complement yeast cell uptake on most tested sugar substrates with a preference for hexoses, while the clade I transporter JsSWEET1 mediates merely galactose import when expressed in yeast. Our results provide first evidence for further investigation on sugar transport and allocation during flowering and reproductive processes in J. sambac.

A new highly sensitive immunoassay for the detection of adiponectin in serum and saliva of dogs and its application in obesity and canine leishmaniosis

Adiponectin is an adipokine that exerts insulin-sensitizing and antiinflammatory properties. The aim of this study was to develop and validate a new heterologous ultrasensitive assay based on amplified luminescent technology for adiponectin determination in serum and saliva of dogs. A complete analytical validation of the assay was made in these fluids, and also this assay was applied to quantify adiponectin in serum and saliva of obese and lean dogs and dogs with leishmaniosis. These conditions were selected because in obesity there is a controversy about how adiponectin concentrations change in dogs, and in case of canine leishmaniosis, although it is described a decrease in serum adiponectin, there are not studies about how adiponectin changes after treatment.A total of 11 dogs were used in the validation and 26 dogs with different body condition and 8 with canine leishmaniosis were used for the clinical evaluation of the new assay for adiponectin quantification in serum and saliva of dogs. The analytical evaluation showed that the developed method could measure adiponectin in serum and saliva of dogs with high repeatability and sensitivity, adding a limit of quantification lower than commercially available ELISAs. In addition, significantly higher adiponectin concentrations were found in lean dogs and a correlation between serum and saliva was observed (P < .01). Moreover, dogs with leishmania presented reduced levels of adiponectin in serum. In conclusion, a new assay has been developed for adiponectin measurements which is more sensitive and faster than the traditional ELISA assays requiring less sample volume.

Characterization of the Copper Transporters from Lotus spp. and Their Involvement under Flooding Conditions.

Forage legumes are an important livestock nutritional resource, which includes essential metals, such as copper. Particularly, the high prevalence of hypocuprosis causes important economic losses to Argentinian cattle agrosystems. Copper deficiency in cattle is partially due to its low content in forage produced by natural grassland, and is exacerbated by flooding conditions. Previous results indicated that incorporation of Lotus spp. into natural grassland increases forage nutritional quality, including higher copper levels. However, the biological processes and molecular mechanisms involved in copper uptake by Lotus spp. remain poorly understood. Here, we identify four genes that encode putative members of the Lotus copper transporter family, denoted COPT in higher plants. A heterologous functional complementation assay of the Saccharomyces cerevisiae ctr1∆ctr3∆ strain, which lacks the corresponding yeast copper transporters, with the putative Lotus COPT proteins shows a partial rescue of the yeast phenotypes in restrictive media. Under partial submergence conditions, the copper content of L. japonicus plants decreases and the expression of two Lotus COPT genes is induced. These results strongly suggest that the Lotus COPT proteins identified in this work function in copper uptake. In addition, the fact that environmental conditions affect the expression of certain COPT genes supports their involvement in adaptive mechanisms and envisages putative biotechnological strategies to improve cattle copper nutrition.

SAT-416 IGSF1 Does Not Regulate FSH Synthesis or Secretion

Loss of function mutations in the human X-linked immunoglobulin superfamily, member 1 (IGSF1) gene result in central hypothyroidism, often associated with macroorchidism. Igsf1-deficient mice are also centrally hypothyroid due to impaired TRH action in the pituitary. The mechanisms underlying macroorchidism are unclear and disputed. IGSF1 was originally characterized as an inhibin B co-receptor. As inhibin B negatively regulates FSH secretion, it was hypothesized that loss of IGSF1 would impair inhibin B action, leading to elevated FSH levels and enhanced Sertoli cell proliferation during development. Yet, in direct contradiction to this model, IGSF1 does not bind inhibin A or B in heterologous binding assays. Our preliminary data further indicate that FSHβ subunit (Fshb) mRNA expression is similarly antagonized by inhibin A and B in pituitary cultures from wild-type and Igsf1-deficient mice. More recently, IGSF1 was proposed to inhibit activin type IB receptor (ALK4) signaling. As activins stimulate FSH, loss of this inhibition should lead to increased FSH. However, neither humans nor mice with IGSF1-deficiency have elevated FSH and IGSF1 expression in FSH-producing gonadotrope cells is negligible. Additionally, Igsf1-deficient mice have normal serum LH and inhibin B levels, as well as testicular weights, histology, and semen parameters. Previous methods used to demonstrate IGSF1 regulation of a human FSHB promoter-reporter were conducted in a heterologous system in which such reporters lack activin/ALK4-dependent activity. We demonstrate that overexpression of IGSF1 in homologous LβT2 gonadotrope-like cells does not impair induction of murine or human Fshb/FSHB promoter-reporters by activin A or a constitutively active form of ALK4. Collectively, the available data fail to support a role for IGSF1 in FSH regulation by activins, inhibins, or otherwise.

Recurrent Germline DLST Mutations in Individuals with Multiple Pheochromocytomas and Paragangliomas

Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and13C5-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (∼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.

Distinct triterpene synthases in the laticifers of Euphorbia lathyris

Euphorbia lathyris was proposed about fifty years ago as a potential agroenergetic crop. The tremendous amounts of triterpenes present in its latex has driven investigations for transforming this particular biological fluid into an industrial hydrocarbon source. The huge accumulation of terpenes in the latex of many plant species represent a challenging question regarding cellular homeostasis. In fact, the enzymes, the mechanisms and the controllers that tune the amount of products accumulated in specialized compartments (to fulfill ecological roles) or deposited at important sites (as essential factors) are not known. Here, we have isolated oxidosqualene cyclases highly expressed in the latex of Euphorbia lathyris. This triterpene biosynthetic machinery is made of distinct paralogous enzymes responsible for the massive accumulation of steroidal and non-steroidal tetracyclic triterpenes. More than eighty years after the isolation of butyrospermol from shea butter (Heilbronn IM, Moffet GL, and Spring FS J. Chem. Soc. 1934, 1583), a butyrospermol synthase is characterized in this work using yeast and in folia heterologous expression assays.