Gene expression and molecular evolution of sxtA4 in a saxitoxin producing dinoflagellate Alexandrium catenella

Copyright © 2014 Elsevier Ltd. All rights reserved. Dinoflagellates of the genus Alexandrium produce the neurotoxin saxitoxin (STX), responsible for paralytic shellfish poisoning (PSP) and accumulates in marine invertebrates. The recent identification of STX biosynthesis genes allowed us to investigate the expression of sxtA4 at different growth stages in Alexandrium catenella Group IV. We found no significant differences in expression of sxtA4, despite significant differences in STX levels at different growth stages (P < 0.023). Three reference genes were tested for normalisation: actin, cytochrome b (cob), and the large subunit ribosomal RNA (LSU rDNA). cob was most stably expressed but the combination of two reference genes, actin and cob, resulted in the best stability factor. Most genomic sequences of sxtA4 from A. catenella were in a clade that included sequences from Alexandrium fundyense Group I, however, one paralogue was not related to the others, suggesting recombination or lateral transfer. A comparison of the sxtA4 cDNA sequences with genomic DNA sequences indicated the possibility of transcript editing and the preferential transcription of certain genomic DNA loci. The results show that, in dinoflagellates, post-transcriptional mechanisms play a major role in the regulation of saxitoxin biosynthesis

Viral communities of Shark Bay modern stromatolites

© 2018 White, Wong, Ruvindy, Neilan and Burns. Single stranded DNA viruses have been previously shown to populate the oceans on a global scale, and are endemic in microbialites of both marine and freshwater systems. We undertook for the first time direct viral metagenomic shotgun sequencing to explore the diversity of viruses in the modern stromatolites of Shark Bay Australia. The data indicate that Shark Bay marine stromatolites have similar diversity of ssDNA viruses to that of Highbourne Cay, Bahamas. ssDNA viruses in cluster uniquely in Shark Bay and Highbourne Cay, potentially due to enrichment by phi29-mediated amplification bias. Further, pyrosequencing data was assembled from the Shark Bay systems into two putative viral genomes that are related to Genomoviridae family of ssDNA viruses. In addition, the cellular fraction was shown to be enriched for antiviral defense genes including CRISPR-Cas, BREX (bacteriophage exclusion), and DISARM (defense island system associated with restriction-modification), a potentially novel finding for these systems. This is the first evidence for viruses in the Shark Bay stromatolites, and these viruses may play key roles in modulating microbial diversity as well as potentially impacting ecosystem function through infection and the recycling of key nutrients

Lack of methylated hopanoids renders the cyanobacterium Nostoc punctiforme sensitive to osmotic and pH stress

© 2017 American Society for Microbiology. To investigate the function of 2-methylhopanoids in modern cyanobacteria, the hpnP gene coding for the radical S-adenosyl methionine (SAM) methylase protein that acts on the C-2 position of hopanoids was deleted from the filamentous cyanobacterium Nostoc punctiforme ATCC 29133S. The resulting ΔhpnP mutant lacked all 2-methylhopanoids but was found to produce much higher levels of two bacteriohopanepentol isomers than the wild type. Growth rates of the ΔhpnP mutant cultures were not significantly different from those of the wild type under standard growth conditions. Akinete formation was also not impeded by the absence of 2-methylhopanoids. The relative abundances of the different hopanoid structures in akinete-dominated cultures of the wild-type and ΔhpnP mutant strains were similar to those of vegetative cell-dominated cultures. However, the ΔhpnP mutant was found to have decreased growth rates under both pH and osmotic stress, confirming a role for 2-methylhopanoids in stress tolerance. Evidence of elevated photosystem II yield and NAD(P)H-dependent oxidoreductase activity in the ΔhpnP mutant under stress conditions, compared to the wild type, suggested that the absence of 2-methylhopanoids increases cellular metabolic rates under stress conditions

A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag)

<p>Abstract</p> <p>Background</p> <p>Genotyping analysis using capillary DNA sequencing with fluorescently labeled primer pairs obtained by polymerase chain reaction (PCR) is widely used, but is expensive. The post-PCR labeling method using fluorescently labeled short oligonucleotides and nested PCR of the amplified product obtained from unlabeled primer pairs is a simple and inexpensive alternative. However, previously reported protocols often produced spurious peaks or inconsistent amplification under multiplexed analysis as a result of simultaneous progress of both the amplification and labeling reactions and local homology of the attached tag sequence.</p> <p>Results</p> <p>A set of 16 bp-long oligonucleotide sequences termed bar-coded split tag (BStag), comprising a common basal region, a three-nucleotide 'bar-code' sequence, and a mismatched nucleotide at the middle position were designed for selective post-PCR labeling. The BStag was attached at the 5' end of the forward primer of interest. The melting temperature of the BStag was low enough to separate the labeling reaction from initial PCR amplification, and each sequence was minimally divergent but maintained maximum selectivity. Post-PCR labeling of the amplified product was achieved by extending for three cycles at a lower annealing temperature after the conventional amplification program with the appropriate fluorescently labeled BStag primer. No amplification was confirmed with BStag primers for 12 plant species. The electropherogram of the labeled product obtained using this method was consistent with that of prelabeled primer, except for their apparent size.</p> <p>Conclusions</p> <p>BStag enabled multiplexed post-PCR labeling of simple sequence repeat or insertion/deletion markers with different dyes in a single tube. BStag in conjunction with locus specific oligo and allele specific oligo was also useful for single nucleotide polymorphism analysis. The labeling protocol was simple and no additional operation was required. Single-tube multiplexed post-PCR labeling is useful for a wide variety of genotyping studies with maximal flexibility and minimal costs.</p

A new species of cryptic cyanobacteria isolated from the epidermis of a bottlenose dolphin and as a bioaerosol

Two cyanobacterial strains, one collected from an epidermal mat present on a dead bottlenose dolphin and the other as a bioaerosol 457 m (1500 ft) above the river, were recently analysed from the St. Johns River, Jacksonville, Florida, USA. Both samples had major phenotypic plasticity which confused morphological identification. Amplicon sequencing of the 16S rRNA gene from the isolates revealed that both samples were closely aligned (branch bootstrap support = 100%) with the recently erected genus Komarekiella. Whole genome sequencing and phylogenetic construction also supported the isolation of a new species of cyanobacteria branching from the Nostoc clade. A total evidence approach of molecular, genetic, and ecological examination of these strains supported the erection of a new species, Komarekiella delphini-convector. A prior study determined that the dolphin with the epidermal mat had low levels of microcystins/nodularins (MCs/NODs) in the hepatic tissue. To investigate whether these toxins originated from the epidermal mat, immunoassay (ELISA) and 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) techniques were conducted on the original mat and subsequent culture samples. The results from both analyses were not conclusive. Genome mining was conducted and revealed diverse biosynthetic capabilities of this species but could not support toxin-producing potential. Further analytical work is required to determine the pathogenic capacity of this epizoic species

Acute epiglottitis as the initial presentation of pediatric Systemic Lupus Erythematosus

We report a case of a 5-year old girl, who initially presented with acute epiglottitis, sepsis and multi-organ failure. She was subsequently diagnosed as having Systemic Lupus Erythematosus. To the best of our knowledge, this article describes the first case of Haemophilus influenzae type f epiglottitis as the initial presentation of SLE in childhood

A canine model of Cohen syndrome: Trapped Neutrophil Syndrome

<p>Abstract</p> <p>Background</p> <p>Trapped Neutrophil Syndrome (TNS) is a common autosomal recessive neutropenia in Border collie dogs.</p> <p>Results</p> <p>We used a candidate gene approach and linkage analysis to show that the causative gene for TNS is <it>VPS13B</it>. We chose <it>VPS13B </it>as a candidate because of similarities in clinical signs between TNS and Cohen syndrome, in human, such as neutropenia and a typical facial dysmorphism. Linkage analysis using microsatellites close to <it>VPS13B </it>showed positive linkage of the region to TNS. We sequenced each of the 63 exons of <it>VPS13B </it>in affected and control dogs and found that the causative mutation in Border collies is a 4 bp deletion in exon 19 of the largest transcript that results in premature truncation of the protein. Cohen syndrome patients present with mental retardation in 99% of cases, but learning disabilities featured in less than half of TNS affected dogs. It has been implied that loss of the alternate transcript of <it>VPS13B </it>in the human brain utilising an alternate exon, 28, may cause mental retardation. Mice cannot be used to test this hypothesis as they do not express the alternate exon. We show that dogs do express alternate transcripts in the brain utilising an alternate exon homologous to human exon 28.</p> <p>Conclusion</p> <p>Dogs can be used as a model organism to explore the function of the alternately spliced transcript of VPS13B in the brain. TNS in Border collies is the first animal model for Cohen syndrome and can be used to study the disease aetiology.</p

Proteome and Membrane Fatty Acid Analyses on Oligotropha carboxidovorans OM5 Grown under Chemolithoautotrophic and Heterotrophic Conditions

Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium able to utilize CO and H2 to derive energy for fixation of CO2. Thus, it is capable of growth using syngas, which is a mixture of varying amounts of CO and H2 generated by organic waste gasification. O. carboxidovorans is capable also of heterotrophic growth in standard bacteriologic media. Here we characterize how the O. carboxidovorans proteome adapts to different lifestyles of chemolithoautotrophy and heterotrophy. Fatty acid methyl ester (FAME) analysis of O. carboxidovorans grown with acetate or with syngas showed that the bacterium changes membrane fatty acid composition. Quantitative shotgun proteomic analysis of O. carboxidovorans grown in the presence of acetate and syngas showed production of proteins encoded on the megaplasmid for assimilating CO and H2 as well as proteins encoded on the chromosome that might have contributed to fatty acid and acetate metabolism. We found that adaptation to chemolithoautotrophic growth involved adaptations in cell envelope, oxidative homeostasis, and metabolic pathways such as glyoxylate shunt and amino acid/cofactor biosynthetic enzymes

Universal Primers Used for Species Identification of Foodstuff of Animal Origin: Effects of Oligonucleotide Tails on PCR Amplification and Sequencing Performance

M13 universal non-homologous oligonucleotide tails incorporated into universal primers have been shown to improve amplification and sequencing performance. However, a few protocols use these tails in the field of food inspection. In this study, two types of M13 tails (by Steffens and Messing) were selected to assess their benefits using universal cytochrome oxidase subunit I (COI) and 16S ribosomal RNA gene (16SrRNA) primers in standard procedures. The primer characteristics were tested in silico. Then, using 20 DNA samples of edible species (birds, fishes, and mammals), their performance during PCR amplification (band recovery and intensity) and sequencing (sequence recovery, length, and Phred score) was assessed and compared. While 16SrRNA tailed and non-tailed primers performed similarly, differences were found for COI primers. Messing’s tails negatively affected the reaction outputs, while Steffens’ tails significantly improved the band intensity and the length of the final contigs based on the individual bidirectional read sequence. This different performance could be related to a destabilization effect of certain tails on primers with unfavorable mismatches on the annealing region. Even though our results cannot be generalized because the tail performances are strictly dependent on laboratory conditions, they show that appropriate tails can improve the overall throughput of the analysis, supporting food traceabilit

Towards accurate and precise T1 and extracellular volume mapping in the myocardium: a guide to current pitfalls and their solutions

Mapping of the longitudinal relaxation time (T1) and extracellular volume (ECV) offers a means of identifying pathological changes in myocardial tissue, including diffuse changes that may be invisible to existing T1-weighted methods. This technique has recently shown strong clinical utility for pathologies such as Anderson- Fabry disease and amyloidosis and has generated clinical interest as a possible means of detecting small changes in diffuse fibrosis; however, scatter in T1 and ECV estimates offers challenges for detecting these changes, and bias limits comparisons between sites and vendors. There are several technical and physiological pitfalls that influence the accuracy (bias) and precision (repeatability) of T1 and ECV mapping methods. The goal of this review is to describe the most significant of these, and detail current solutions, in order to aid scientists and clinicians to maximise the utility of T1 mapping in their clinical or research setting. A detailed summary of technical and physiological factors, issues relating to contrast agents, and specific disease-related issues is provided, along with some considerations on the future directions of the field. Towards accurate and precise T1 and extracellular volume mapping in the myocardium: a guide to current pitfalls and their solutions. Available from: https://www.researchgate.net/publication/317548806_Towards_accurate_and_precise_T1_and_extracellular_volume_mapping_in_the_myocardium_a_guide_to_current_pitfalls_and_their_solutions [accessed Jun 13, 2017]