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September 22, 2019

Effects of low crude oil chronic exposure on the northern krill (Meganyctiphanes norvegica)

Chronic oil pollution related to gas and oil drilling activities is increasing in the sea due to the rising offshore petroleum industry activity. Among marine organisms, zooplankton play a crucial role in the marine ecosystem and therefore understanding the effects of crude oil chronic exposure on zooplankton is needed to determine the impact of oil in marine environments. The present study reports on the effect of crude oil on adult northern krill, Meganyctiphanes norvegica, collected during three seasons. Their sensitivity to oil was examined with oil concentration of 0.01 versus 0.1 mg oil L- 1 and photo-modified oil in flowing seawater maintained in the dark for 2 weeks at in situ temperature. Oil (polycyclic aromatic hydrocarbons, PAHs) entered the krill (on average, 350 and 4400 µg·kg- 1 wet weight in low and medium oil treatments respectively) and a larger fraction of the krill exhibited digestive gland pathologies (enhanced apoptosis and pathology of digestive tubules) in oil treatments (27–80%) compared to a significantly lower fraction (7–13%) in treatments that received no oil. However, 2-week oil exposure at these concentrations did not significantly decrease survivorship or impair basic functioning such as feeding and respiration rates. Similarly, there were only limited changes in the transcription of 7 selected genes from head tissue. Additionally, although there was significant seasonal variation in krill total lipid content and fatty acid composition, there was no treatment effect on both these parameters, which suggests limited oxidative stress under experimental conditions. Furthermore, there was no significant treatment effect on two direct measures of oxidative stress (MDA: malondialdehyde and AOPP: advanced oxidation protein products) in any of the seasons. Nevertheless, histology clearly revealed enhanced digestive gland pathologies in krill even at low concentrations. Although krill with such pathologies continue to survive, their accumulation of PAHs may be transferred up the food chain, impacting their predators and the wider ecosystem.

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September 22, 2019

Meeting report: 31st International Mammalian Genome Conference, Mammalian Genetics and Genomics: From Molecular Mechanisms to Translational Applications.

High on the Heidelberg hills, inside the Advanced Training Centre of the European Molecular Biology Laboratory (EMBL) campus with its unique double-helix staircase, scientists gathered for the EMBL conference “Mammalian Genetics and Genomics: From Molecular Mechanisms to Translational Applications,” organized in cooperation with the International Mammalian Genome Society (IMGS) and the Mouse Molecular Genetics (MMG) group. The conference attracted 205 participants from 30 countries, representing 6 of the 7 continents-all except Antarctica. It was a richly diverse group of geneticists, clinicians, and bioinformaticians, with presentations by established and junior investigators, including many trainees. From the 24th-27th of October 2017, they shared exciting advances in mammalian genetics and genomics research, from the introduction of cutting-edge technologies to descriptions of translational studies involving highly relevant models of human disease.

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September 22, 2019

Young genes have distinct gene structure, epigenetic profiles, and transcriptional regulation.

Species-specific, new, or “orphan” genes account for 10%-30% of eukaryotic genomes. Although initially considered to have limited function, an increasing number of orphan genes have been shown to provide important phenotypic innovation. How new genes acquire regulatory sequences for proper temporal and spatial expression is unknown. Orphan gene regulation may rely in part on origination in open chromatin adjacent to preexisting promoters, although this has not yet been assessed by genome-wide analysis of chromatin states. Here, we combine taxon-rich nematode phylogenies with Iso-Seq, RNA-seq, ChIP-seq, and ATAC-seq to identify the gene structure and epigenetic signature of orphan genes in the satellite model nematode Pristionchus pacificus Consistent with previous findings, we find young genes are shorter, contain fewer exons, and are on average less strongly expressed than older genes. However, the subset of orphan genes that are expressed exhibit distinct chromatin states from similarly expressed conserved genes. Orphan gene transcription is determined by a lack of repressive histone modifications, confirming long-held hypotheses that open chromatin is important for new gene formation. Yet orphan gene start sites more closely resemble enhancers defined by H3K4me1, H3K27ac, and ATAC-seq peaks, in contrast to conserved genes that exhibit traditional promoters defined by H3K4me3 and H3K27ac. Although the majority of orphan genes are located on chromosome arms that contain high recombination rates and repressive histone marks, strongly expressed orphan genes are more randomly distributed. Our results support a model of new gene origination by rare integration into open chromatin near enhancers.© 2018 Werner et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019

A community-based culture collection for targeting novel plant growth-promoting bacteria from the sugarcane microbiome.

The soil-plant ecosystem harbors an immense microbial diversity that challenges investigative approaches to study traits underlying plant-microbe association. Studies solely based on culture-dependent techniques have overlooked most microbial diversity. Here we describe the concomitant use of culture-dependent and -independent techniques to target plant-beneficial microbial groups from the sugarcane microbiome. The community-based culture collection (CBC) approach was used to access microbes from roots and stalks. The CBC recovered 399 unique bacteria representing 15.9% of the rhizosphere core microbiome and 61.6-65.3% of the endophytic core microbiomes of stalks. By cross-referencing the CBC (culture-dependent) with the sugarcane microbiome profile (culture-independent), we designed a synthetic community comprised of naturally occurring highly abundant bacterial groups from roots and stalks, most of which has been poorly explored so far. We then used maize as a model to probe the abundance-based synthetic inoculant. We show that when inoculated in maize plants, members of the synthetic community efficiently colonize plant organs, displace the natural microbiota and dominate at 53.9% of the rhizosphere microbial abundance. As a result, inoculated plants increased biomass by 3.4-fold as compared to uninoculated plants. The results demonstrate that abundance-based synthetic inoculants can be successfully applied to recover beneficial plant microbes from plant microbiota.

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September 22, 2019

Comparative transcriptome analysis of genes involved in Na+ transport in the leaves of halophyte Halogeton glomeratus.

Compartmentalization of Na+ into vacuoles is considered to be the most critical aspect of salt tolerance in H. glomeratus, an annual, succulent halophyte. Previous analysis of transcriptome involved in the H. glomeratus salt stress response relied on next-generation sequencing technologies that limit the capture of accurately spliced, full-length isoforms. To gain deeper insights into its salt stress response, we used the H. glomeratus Iso-Seq transcriptome database as a reference, and subsequent next-generation sequencing was subjected to various NaCl concentrations of leaves from plants revealed 115 upregulated and 87 downregulated differentially expressed isoforms (core DEIs). The majority of the core DEIs were involved in carbohydrate metabolism and energy production and conversion. In contrast, levels of known isoforms encoding Na+ transporters did not change significantly under salt stress. However, 16 core DEIs of unknown function were predicted to possess transmembrane domains, suggesting that these candidate isoforms could be involved in Na+ transport in H. glomeratus. These results suggest a potential means for identification of novel Na+ transporters, in addition to providing a foundation for further investigation of Na+ transport networks in halophytes. Copyright © 2018. Published by Elsevier B.V.

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September 22, 2019

Automated broad range molecular detection of bacteria in clinical samples.

Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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September 22, 2019

Bacteroides dorei dominates gut microbiome prior to autoimmunity in Finnish children at high risk for type 1 diabetes.

The incidence of the autoimmune disease, type 1 diabetes (T1D), has increased dramatically over the last half century in many developed countries and is particularly high in Finland and other Nordic countries. Along with genetic predisposition, environmental factors are thought to play a critical role in this increase. As with other autoimmune diseases, the gut microbiome is thought to play a potential role in controlling progression to T1D in children with high genetic risk, but we know little about how the gut microbiome develops in children with high genetic risk for T1D. In this study, the early development of the gut microbiomes of 76 children at high genetic risk for T1D was determined using high-throughput 16S rRNA gene sequencing. Stool samples from children born in the same hospital in Turku, Finland were collected at monthly intervals beginning at 4-6 months after birth until 2.2 years of age. Of those 76 children, 29 seroconverted to T1D-related autoimmunity (cases) including 22 who later developed T1D, the remaining 47 subjects remained healthy (controls). While several significant compositional differences in low abundant species prior to seroconversion were found, one highly abundant group composed of two closely related species, Bacteroides dorei and Bacteroides vulgatus, was significantly higher in cases compared to controls prior to seroconversion. Metagenomic sequencing of samples high in the abundance of the B. dorei/vulgatus group before seroconversion, as well as longer 16S rRNA sequencing identified this group as Bacteroides dorei. The abundance of B. dorei peaked at 7.6 months in cases, over 8 months prior to the appearance of the first islet autoantibody, suggesting that early changes in the microbiome may be useful for predicting T1D autoimmunity in genetically susceptible infants. The cause of increased B. dorei abundance in cases is not known but its timing appears to coincide with the introduction of solid food.

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September 22, 2019

Investigating bacterial population structure and dynamics in traditional koumiss from Inner Mongolia using single molecule real-time sequencing.

Koumiss is considered as a complete dairy product high in nutrients and with medicinal properties. The bacterial communities involved in production of koumiss play a crucial role in the fermentation cycle. To reveal bacterial biodiversity in koumiss and the dynamics of succession in bacterial populations during fermentation, 22 samples were collected from 5 sampling sites and the full length of the 16S ribosomal RNA genes sequenced using single molecule real-time sequencing technology. One hundred forty-eight species were identified from 82 bacterial genera and 8 phyla. These results suggested that the structural difference in the bacterial community could be attributed to geographical location. The most significant difference in bacterial composition occurred in samples from group D compared with other groups. The sampling location of group D was distant from the city and maintained the primitive local nomadic life. The dynamics of succession in bacterial communities showed that Lactobacillus helveticus increased in abundance from 0 to 9h and reached its peak at 9h and then decreased. In contrast, Enterococcus faecalis, Enterococcus durans, and Enterococcus casseliflavus increased gradually throughout the fermentation process, and reached a maximum after 24h. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Analyses of intestinal microbiota: culture versus sequencing.

Analyzing human as well as animal microbiota composition has gained growing interest because structural components and metabolites of microorganisms fundamentally influence all aspects of host physiology. Originally dominated by culture-dependent methods for exploring these ecosystems, the development of molecular techniques such as high throughput sequencing has dramatically increased our knowledge. Because many studies of the microbiota are based on the bacterial 16S ribosomal RNA (rRNA) gene targets, they can, at least in principle, be compared to determine the role of the microbiome composition for developmental processes, host metabolism, and physiology as well as different diseases. In our review, we will summarize differences and pitfalls in current experimental protocols, including all steps from nucleic acid extraction to bioinformatical analysis which may produce variation that outweighs subtle biological differences. Future developments, such as integration of metabolomic, transcriptomic, and metagenomic data sets and standardization of the procedures, will be discussed. © The Author 2015. Published by Oxford University Press on behalf of the Institute for Laboratory Animal Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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September 22, 2019

Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia

Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein levels of AID splice isoforms by introducing an AID splice reporter construct into cell lines and primary CLL cells from patients as well as from WT and TCL1(tg) C57BL/6 mice (where TCL1 is T-cell leukemia/lymphoma 1). The splice construct is 5′-fused to a GFP-tag, which is preserved in all splice isoforms and allows detection of translated protein. Summarizing, we show a thorough quantification of alternatively spliced AID transcripts and demonstrate that the corresponding protein abundances, especially those of splice variants AID-ivs3 and AID-?E4, are not stoichiometrically equivalent. Our data suggest that enhanced proteasomal degradation of low-abundance proteins might be causative for this discrepancy. © 2013 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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September 22, 2019

Molecular mechanisms of acclimatization to phosphorus starvation and recovery underlying full-length transcriptome profiling in barley (Hordeum vulgare L.).

A lack of phosphorus (P) in plants can severely constrain growth and development. Barley, one of the earliest domesticated crops, is extensively planted in poor soil around the world. To date, the molecular mechanisms of enduring low phosphorus, at the transcriptional level, in barley are still unclear. In the present study, two different barley genotypes (GN121 and GN42)-with contrasting phosphorus efficiency-were used to reveal adaptations to low phosphorus stress, at three time points, at the morphological, physiological, biochemical, and transcriptome level. GN121 growth was less affected by phosphorus starvation and recovery than that of GN42. The biomass and inorganic phosphorus concentration of GN121 and GN42 declined under the low phosphorus-induced stress and increased after recovery with normal phosphorus. However, the range of these parameters was higher in GN42 than in GN121. Subsequently, a more complete genome annotation was obtained by correcting with the data sequenced on Illumina HiSeq X 10 and PacBio RSII SMRT platform. A total of 6,182 and 5,270 differentially expressed genes (DEGs) were identified in GN121 and GN42, respectively. The majority of these DEGs were involved in phosphorus metabolism such as phospholipid degradation, hydrolysis of phosphoric enzymes, sucrose synthesis, phosphorylation/dephosphorylation and post-transcriptional regulation; expression of these genes was significantly different between GN121 and GN42. Specifically, six and seven DEGs were annotated as phosphorus transporters in roots and leaves, respectively. Furthermore, a putative model was constructed relying on key metabolic pathways related to phosphorus to illustrate the higher phosphorus efficiency of GN121 compared to GN42 under low phosphorus conditions. Results from this study provide a multi-transcriptome database and candidate genes for further study on phosphorus use efficiency (PUE).

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September 22, 2019

Somatic APP gene recombination in Alzheimer’s disease and normal neurons.

The diversity and complexity of the human brain are widely assumed to be encoded within a constant genome. Somatic gene recombination, which changes germline DNA sequences to increase molecular diversity, could theoretically alter this code but has not been documented in the brain, to our knowledge. Here we describe recombination of the Alzheimer’s disease-related gene APP, which encodes amyloid precursor protein, in human neurons, occurring mosaically as thousands of variant ‘genomic cDNAs’ (gencDNAs). gencDNAs lacked introns and ranged from full-length cDNA copies of expressed, brain-specific RNA splice variants to myriad smaller forms that contained intra-exonic junctions, insertions, deletions, and/or single nucleotide variations. DNA in situ hybridization identified gencDNAs within single neurons that were distinct from wild-type loci and absent from non-neuronal cells. Mechanistic studies supported neuronal ‘retro-insertion’ of RNA to produce gencDNAs; this process involved transcription, DNA breaks, reverse transcriptase activity, and age. Neurons from individuals with sporadic Alzheimer’s disease showed increased gencDNA diversity, including eleven mutations known to be associated with familial Alzheimer’s disease that were absent from healthy neurons. Neuronal gene recombination may allow ‘recording’ of neural activity for selective ‘playback’ of preferred gene variants whose expression bypasses splicing; this has implications for cellular diversity, learning and memory, plasticity, and diseases of the human brain.

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September 22, 2019

Resistance to ceftazidime-avibactam in Klebsiella pneumoniae due to porin mutations and the increased expression of KPC-3.

We reported the first clinical case of a ceftazidime-avibactam resistant KPC-3-producing Klebsiella pneumoniae (1), from a patient with no history of ceftazidime-avibactam therapy. We now present data documenting mechanisms of ceftazidime-avibactam resistance in this isolate. Whole-genome sequencing (WGS) was performed on two isolates: KP1245 (ceftazidime-avibactam MIC, 4 µg/ml; from blood on hospital day 1; referred to as isolate 1 in our previous report [1]) and KP1244 (ceftazidime-avibactam MIC, 32 µg/ml; from blood on hospital day 2; referred to as isolate 2 in our previous report [2]), using MiSeq (Illumina, San Diego, CA) and PacBio RSII (Menlo Park, CA) systems (2). The in silico multilocus sequence type (ST) was ST258. Single nucleotide polymorphism (SNP) analysis revealed 17 SNPs between KP1245 and KP1244, indicating that the isolates were related but that significant diversity existed in this patient (2). Nonsynonymous mutations are shown in Table 1; the most striking of these is in the OmpK36 porin gene. KP1244 contained a missense mutation predicted to encode a T333N mutation. Both isolates also harbored a mutation predicted to encode R191L in OmpK36 and had a nonfunctional OmpK35, due to a frameshift mutation that truncated the protein at amino acid 42, common to K. pneumoniae ST258 (3). Association between mutations in ompK36 and elevated ceftazidime-avibactam MICs has been shown previously (4). However, T333N, found in one of the ß-sheet domains of the OmpK36 subunit, has not been described in K. pneumoniae; as such, further validation is required to confirm the role of the OmpK36 mutation in this isolate’s ceftazidime-avibactam resistance phenotype.

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September 22, 2019

Quantitative profiling of Drosophila melanogaster Dscam1 isoforms reveals no changes in splicing after bacterial exposure.

The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first time, use a long read RNA sequencing method to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore our studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens.

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September 22, 2019

Active microorganisms in forest soils differ from the total community yet are shaped by the same environmental factors: the influence of pH and soil moisture.

Predicting the impact of environmental change on soil microbial functions requires an understanding of how environmental factors shape microbial composition. Here, we investigated the influence of environmental factors on bacterial and fungal communities across an expanse of northern hardwood forest in Michigan, USA, which spans a 500-km regional climate gradient. We quantified soil microbial community composition using high-throughput DNA sequencing on coextracted rDNA (i.e. total community) and rRNA (i.e. active community). Within both bacteria and fungi, total and active communities were compositionally distinct from one another across the regional gradient (bacteria P = 0.01; fungi P < 0.01). Taxonomically, the active community was a subset of the total community. Compositional differences between total and active communities reflected changes in the relative abundance of dominant taxa. The composition of both the total and active microbial communities varied by site across the gradient (P < 0.01) and was shaped by differences in soil moisture, pH, SOM carboxyl content, as well as C and N concentration. Our study highlights the importance of distinguishing between metabolically active microorganisms and the total community, and emphasizes that the same environmental factors shape the total and active communities of bacteria and fungi in this ecosystem.© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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