D27-like carotenoid isomerases: at the crossroads of strigolactone and abscisic acid biosynthesis.

Strigolactones and abscisic acid (ABA) are apocarotenoid-derived plant hormones. Their biosynthesis starts with the conversion of trans-carotenes into cis forms, which serve as direct precursors. Iron-containing DWARF27 isomerases were shown to catalyse or contribute to the trans/cis conversions of these precursor molecules. D27 converts trans-ß-carotene into 9-cis-ß-carotene, which is the first committed step in strigolactone biosynthesis. Recent studies found that its paralogue, D27-LIKE1, also catalyses this conversion. A crucial step in ABA biosynthesis is the oxidative cleavage of 9-cis-violaxanthin and/or 9-cis-neoxanthin, which are formed from their trans isomers by unknown isomerases. Several lines of evidence point out that D27-like proteins directly or indirectly contribute to 9-cis-violaxanthin conversion, and eventually ABA biosynthesis. Apparently, the diversity of D27-like enzymatic activity is essential for the optimization of cis/trans ratios, and hence act to maintain apocarotenoid precursor pools. In this review, we discuss the functional divergence and redundancy of D27 paralogues and their potential direct contribution to ABA precursor biosynthesis. We provide updates on their gene expression regulation and alleged Fe-S cluster binding feature. Finally, we conclude that the functional divergence of these paralogues is not fully understood and we provide an outlook on potential directions in research.

Tryptophan-like side chain holding aptamers inhibit respiratory syncytial virus infection of lung epithelial cells.

Respiratory syncytial virus (RSV) is a leading cause of serious and even fatal acute lower respiratory tract infections in infants and in the elderly. Potent RSV neutralization has been achieved by antibodies that selectively bind the prefusion form of the viral fusion (F) protein. We hypothesised that similar potent neutralization could be achieved using F protein targeting aptamers. Aptamers have yet to reach their translational potential for therapeutics or diagnostics due to their short half-life and limited range of target-aptamer interactions; these shortcomings can, however, be ameliorated by application of amino acid-like side chain holding nucleotides. In this study, a stabilized version of the prefusion RSV F protein was targeted by aptamer selection using an oligonucleotide library holding a tryptophan-like side chain. This process resulted in aptamers that bound the F protein with high affinity and differentiated between its pre- and postfusion conformation. Identified aptamers inhibited viral infection of lung epithelial cells. Moreover, introduction of modified nucleotides extended aptamer half-lives. Our results suggest that targeting aptamers to the surface of viruses could yield effective drug candidates, which could keep pace with the continuously evolving pathogens.

D27-LIKE1 isomerase has a preference towards trans/cis and cis/cis conversions of carotenoids in Arabidopsis.

Carotenoids contribute to a variety of physiological processes in plants, functioning also as biosynthesis precursors of ABA and strigolactones (SLs). SL biosynthesis starts with the enzymatic conversion of all-trans-ß-carotene to 9-cis-ß-carotene by the DWARF27 (D27) isomerase. In Arabidopsis, D27 has two closely related paralogs, D27-LIKE1 and D27-LIKE2, which were predicted to be ß-carotene-isomerases. In the present study, we characterised D27-LIKE1 and identified some key aspects of its physiological and enzymatic functions in Arabidopsis. d27-like1-1 mutant does not display any strigolactone-deficient traits and exhibits a substantially higher 9-cis-violaxanthin content, which is accompanied by a slightly higher ABA level. In vitro feeding assays with recombinant D27-LIKE1 revealed that the protein exhibits affinity to all ß-carotene isoforms but with an exclusive preference towards trans/cis conversions and the interconversion between 9-cis, 13-cis and 15-cis-ß-carotene forms, and accepts zeaxanthin and violaxanthin as substrates. Finally, we present evidence showing that D27-LIKE1 mRNA is phloem mobile and D27-LIKE1 is an ancient isomerase with a long evolutionary history. In summary, we demonstrate that D27-LIKE1 is a carotenoid isomerase with multi-substrate specificity and has a characteristic preference towards the catalysation of cis/cis interconversion of carotenoids. Therefore, D27-LIKE1 is a potential regulator of carotenoid cis pools and, eventually, SL and ABA biosynthesis pathways.

Emerging human alveolar echinococcosis in Hungary (2003-2018): a retrospective case series analysis from a multi-centre study.

BACKGROUND: Human alveolar echinococcosis (AE) caused by Echinococcus multilocularis is an underreported, often misdiagnosed and mistreated parasitic disease mainly due to its low incidence. The aim of this study was to describe the epidemiological and clinical characteristics of human AE patients in Hungary for the first time. METHOD: Between 2003 and 2018, epidemiological and clinical data of suspected AE patients were collected retrospectively from health database management systems. RESULTS: This case series included a total of 16 AE patients. The mean age of patients was 53 years (range: 24-78 years). The sex ratio was 1:1. Four patients (25%) revealed no recurrence after radical surgery and adjuvant albendazole (ABZ) therapy. For five patients (31.3%) with unresectable lesions, a stabilization of lesions with ABZ treatment was achieved. In seven patients (43.8%), progression of AE was documented. The mean diagnostic delay was 33 months (range: 1-122 months). Three AE related deaths (fatality rate 18.8%) were recorded. CONCLUSIONS: AE is an emerging infectious disease in Hungary with a high fatality rate since based on our results, almost every fifth AE patient died in the study period. Differential diagnosis and appropriate surgical and medical therapy for AE is an urging challenge for clinicians in Hungary, as well as in some other European countries where E. multilocularis is prevalent.

Spiegelmer-Based Sandwich Assay for Cardiac Troponin I Detection.

Two subunits of the ternary troponin complex, I and C, have cardiac muscle specific isoforms, and hence could be applied as highly-selective markers of acute coronary syndrome. We aimed at paving the way for the development of a robust cardiac troponin I-detecting sandwich assay by replacing antibodies with nuclease resistant aptamer analogues, so-called spiegelmers. To complement the previously generated spiegelmers that were specific for the N-terminus of cTnI, spiegelmers were selected for an amino acid stretch in the proximity of the C-terminal part of the protein by using a D-amino acid composed peptide. Following the selection, the oligonucleotides were screened by filter binding assay, and surface plasmon resonance analysis of the most auspicious candidates demonstrated that this approach could provide spiegelmers with subnanomolar dissociation constant. To demonstrate if the selected spiegelmers are functional and suitable for cTnI detection in a sandwich type arrangement, AlphaLisa technology was leveraged and the obtained results demonstrated that spiegelmers with different epitope selectivity are suitable for specific detection of cTnI protein even in human plasma containing samples. These results suggest that spiegelmers could be considered in the development of the next generation cTnI monitoring assays.

Rapid spread and emergence of heartworm resulting from climate and climate-driven ecological changes in Hungary.

In Europe, Dirofilaria immitis persists mainly in the southern countries with a Mediterranean climate. Because spreading of heartworms from these countries towards the northern ones could be observed in the past decades, necropsy records of 4076 Hungarian dogs were reviewed for heartworm infections. The first autochthonous canine D. immitis case was detected on the Great Hungarian Plain in 2007. Until 2011, the number of heartworm infection cases was low, and these cases were restricted to a small part of the Great Hungarian Plain. Since 2012, the number of cases has increased considerably, and the rapid expansion of the parasite's geographic range could also be observed. Our retrospective study has revealed that most of the Hungarian territory became a heartworm endemic region, and the prevalence of infection greatly multiplied over the past 12 years. The establishment, rapid spread, and emergence of D. immitis may be mainly explained by the warming climate in Hungary. However, the partly climate-driven spread of the most important reservoir host in wildlife, the golden jackal (Canis aureus) from the Mediterranean Balkan Peninsula might have also played a significant role. This study is an example of the rapid spread and emergence of pathogens resulting from climate and climate-driven ecological changes. Because a continuous increase in the temperature and further dispersal of golden jackals in Europe are projected, further spread and emergence of heartworm can be expected. Similar spread and emergence of D. immitis could be observed in North America. It cannot be excluded that similar reasons (global warming and rapid dispersal and population growth of the most important wild canine reservoir host) are in the background on both continents.

A simple modification increases specificity and efficiency of asymmetric PCR.

Although various methods have been developed to suffice the oligonucleotide demand of molecular biology laboratories, in vitro production of high-purity ssDNAs remains to be a challenging task. We hypothesized that complementing the asymmetric PCR with 3' phosphate blocked limiting primer decreases the mispriming thus reduces polymerisation of DNA by-products. The presented results attest our assumption that the primer blocked asymmetric PCR (PBA-PCR) selectively produces ssDNA of interest and is even suitable for effective amplification of DNA libraries of large sequence space. The high-throughput sequence analysis demonstrated that PBA-PCR also alleviates the PCR bias obstacle since it does not distort the sequence space. The practicability of the novel method was verified by monitoring the process of SELEX and screening of aptamer candidates using PBA-PCR produced ssDNAs in Amplified Luminescent Proximity Homogeneous Assay. In summary, we have developed a generally applicable method for straightforward, cost-effective production of ssDNA with on demand labelling.

Parental genetic diversity of brown trout (Salmo trutta m. fario) brood stock affects offspring susceptibility to whirling disease.

BACKGROUND: Whirling disease, caused by the myxozoan parasite Myxobolus cerebralis, has high economical and ecological importance worldwide. Susceptibility to the disease varies considerably among salmonid species. In brown trout (Salmo trutta) the infection is usually subclinical with low mortality, which increases the risk of parasite dissemination, especially when farm fish are used for stocking natural habitats. The influence of intraspecific genetic differences (especially the level of homozygosity) on susceptibility is unknown. Therefore, we examined the possible correlations between parental genetic diversity and offspring susceptibility of brown trout stocks to whirling disease. METHODS: Two brown trout brood stocks from a German and a Hungarian fish farm were genetically characterized using microsatellite and lineage-specific genetic markers. The individual inbreeding coefficient f and pairwise relatedness factor r were estimated based on eight microsatellite markers. Brood stock populations were divided into groups according to low and high f and r value estimates and subjected to selective fertilization. The offspring from these separate groups were exposed to M. cerebralis actinospores, and the infection prevalence and intensity was measured and statistically analysed. RESULTS: The analysis of phylogeographic lineage heritage revealed high heterogeneity in the Hungarian brood stock since > 50% of individuals were Atlantic-Danubian hybrids, while only pure Atlantic-descending specimens were detected in the German population. Based on f msat and r msat estimations, classified non-inbred (NIB), inbred (IB) and a group of closely related fish (REL) were created. The susceptibility of their offspring varied considerably. Although there was no significant difference in the prevalence of M. cerebralis infection, the mean intensity of infection differed significantly between NIB and IB groups. In REL and IB groups, a high variability was observed in infection intensity. No external clinical signs were observed in the exposed brown trout groups. CONCLUSIONS: Our findings indicate that the allelic diversity of brown trout brood stock may constitute a significant factor in disease susceptibility, i.e. the intensity of parasite infection in the subsequent generation.