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September 22, 2019

Gut microbiota, nitric oxide, and microglia as prerequisites for neurodegenerative disorders.

Regulating fluctuating endogenous nitric oxide (NO) levels is necessary for proper physiological functions. Aberrant NO pathways are implicated in a number of neurological disorders, including Alzheimer’s disease (AD) and Parkinson’s disease. The mechanism of NO in oxidative and nitrosative stress with pathological consequences involves reactions with reactive oxygen species (e.g., superoxide) to form the highly reactive peroxynitrite, hydrogen peroxide, hypochloride ions and hydroxyl radical. NO levels are typically regulated by endogenous nitric oxide synthases (NOS), and inflammatory iNOS is implicated in the pathogenesis of neurodegenerative diseases, in which elevated NO mediates axonal degeneration and activates cyclooxygenases to provoke neuroinflammation. NO also instigates a down-regulated secretion of brain-derived neurotrophic factor, which is essential for neuronal survival, development and differentiation, synaptogenesis, and learning and memory. The gut-brain axis denotes communication between the enteric nervous system (ENS) of the GI tract and the central nervous system (CNS) of the brain, and the modes of communication include the vagus nerve, passive diffusion and carrier by oxyhemoglobin. Amyloid precursor protein that forms amyloid beta plaques in AD is normally expressed in the ENS by gut bacteria, but when amyloid beta accumulates, it compromises CNS functions. Escherichia coli and Salmonella enterica are among the many bacterial strains that express and secrete amyloid proteins and contribute to AD pathogenesis. Gut microbiota is essential for regulating microglia maturation and activation, and activated microglia secrete significant amounts of iNOS. Pharmacological interventions and lifestyle modifications to rectify aberrant NO signaling in AD include NOS inhibitors, NMDA receptor antagonists, potassium channel modulators, probiotics, diet, and exercise.

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September 22, 2019

Transgenerational attenuation of opioid self-administration as a consequence of adolescent morphine exposure.

The United States is in the midst of an opiate epidemic, with abuse of prescription and illegal opioids increasing steadily over the past decade. While it is clear that there is a genetic component to opioid addiction, there is a significant portion of heritability that cannot be explained by genetics alone. The current study was designed to test the hypothesis that maternal exposure to opioids prior to pregnancy alters abuse liability in subsequent generations. Female adolescent Sprague Dawley rats were administered morphine at increasing doses (5-25 mg/kg, s.c.) or saline for 10 days (P30-39). During adulthood, animals were bred with drug-naïve colony males. Male and female adult offspring (F1 animals) were tested for morphine self-administration acquisition, progressive ratio, extinction, and reinstatement at three doses of morphine (0.25, 0.75, 1.25 mg/kg/infusion). Grand offspring (F2 animals, from the maternal line) were also examined. Additionally, gene expression changes within the nucleus accumbens were examined with RNA deep sequencing (PacBio) and qPCR. There were dose- and sex-dependent effects on all phases of the self-administration paradigm that indicate decreased morphine reinforcement and attenuated relapse-like behavior. Additionally, genes related to synaptic plasticity, as well as myelin basic protein (MBP), were dysregulated. Some, but not all, effects persisted into the subsequent (F2) generation. The results demonstrate that even limited opioid exposure during adolescence can have lasting effects across multiple generations, which has implications for mechanisms of the transmission of drug abuse liability in humans. Copyright © 2016 Elsevier Ltd. All rights reserved.

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September 22, 2019

Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity.

Circular RNAs (circRNAs) have re-emerged as an interesting RNA species. Using deep RNA profiling in different mouse tissues, we observed that circRNAs were substantially enriched in brain and a disproportionate fraction of them were derived from host genes that encode synaptic proteins. Moreover, on the basis of separate profiling of the RNAs localized in neuronal cell bodies and neuropil, circRNAs were, on average, more enriched in the neuropil than their host gene mRNA isoforms. Using high-resolution in situ hybridization, we visualized circRNA punctae in the dendrites of neurons. Consistent with the idea that circRNAs might regulate synaptic function during development, many circRNAs changed their abundance abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibited substantial up- or downregulation. Together, our data indicate that brain circRNAs are positioned to respond to and regulate synaptic function.

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September 22, 2019

Next-generation sequencing for pathogen detection and identification

Over the past decade, the field of genomics has seen such drastic improvements in sequencing chemistries that high-throughput sequencing, or next-generation sequencing (NGS), is being applied to generate data across many disciplines. NGS instruments are becoming less expensive, faster, and smaller, and therefore are being adopted in an increasing number of laboratories, including clinical laboratories. Thus far, clinical use of NGS has been mostly focused on the human genome, for purposes such as characterizing the molecular basis of cancer or for diagnosing and understanding the basis of rare genetic disorders. There are, however, an increasing number of examples whereby NGS is employed to discover novel pathogens, and these cases provide precedent for the use of NGS in microbial diagnostics. NGS has many advantages over traditional microbial diagnostic methods, such as unbiased rather than pathogen-specific protocols, ability to detect fastidious or non-culturable organisms, and ability to detect co-infections. One of the most impressive advantages of NGS is that it requires little or no prior knowledge of the pathogen, unlike many other diagnostic assays; therefore for pathogen discovery, NGS is very valuable. However, despite these advantages, there are challenges involved in implementing NGS for routine clinical microbiological diagnosis. We discuss these advantages and challenges in the context of recently described research studies.

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September 22, 2019

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019

Next-generation approaches to advancing eco-immunogenomic research in critically endangered primates.

High-throughput sequencing platforms are generating massive amounts of genomic data from nonmodel species, and these data sets are valuable resources that can be mined to advance a number of research areas. An example is the growing amount of transcriptome data that allow for examination of gene expression in nonmodel species. Here, we show how publicly available transcriptome data from nonmodel primates can be used to design novel research focused on immunogenomics. We mined transcriptome data from the world’s most endangered group of primates, the lemurs of Madagascar, for sequences corresponding to immunoglobulins. Our results confirmed homology between strepsirrhine and haplorrhine primate immunoglobulins and allowed for high-throughput sequencing of expressed antibodies (Ig-seq) in Coquerel’s sifaka (Propithecus coquereli). Using both Pacific Biosciences RS and Ion Torrent PGM sequencing, we performed Ig-seq on two individuals of Coquerel’s sifaka. We generated over 150 000 sequences of expressed antibodies, allowing for molecular characterization of the antigen-binding region. Our analyses suggest that similar VDJ expression patterns exist across all primates, with sequences closely related to the human VH 3 immunoglobulin family being heavily represented in sifaka antibodies. Moreover, the antigen-binding region of sifaka antibodies exhibited similar amino acid variation with respect to haplorrhine primates. Our study represents the first attempt to characterize sequence diversity of the expressed antibody repertoire in a species of lemur. We anticipate that methods similar to ours will provide the framework for investigating the adaptive immune response in wild populations of other nonmodel organisms and can be used to advance the burgeoning field of eco-immunology. © 2014 John Wiley & Sons Ltd.

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September 22, 2019

Identification of putative coffee rust mycoparasites using single molecule DNA sequencing of infected pustules.

The interaction of crop pests with their natural enemies is a fundament to their control. Natural enemies of fungal pathogens of crops are poorly known relative to those of insect pests despite the diversity of fungal pathogens and their economic importance. Currently, many regions across Latin America are experiencing unprecedented epidemics of coffee rust (Hemileia vastatrix). Identification of natural enemies of coffee rust could aid in developing management strategies or in pinpointing species that could be used for biocontrol. Here we characterize fungal communities associated with coffee rust lesions by single molecule DNA sequencing of fungal ribosomal RNA barcodes from leaf discs (˜28 mm(2)) containing rust lesions and control discs with no rust lesions. The leaf disc communities were hyper-diverse in fungi, with up to 57 taxa per control disc, and the diversity was only slightly reduced in rust-infected discs. However, geography had a greater influence on the fungal community than whether the disk was infected by coffee rust. Through comparisons between control and rust-infected leaf discs, as well as taxonomic criteria, we identified 15 putative mycoparasitic fungi. These fungi are concentrated in fungal family Cordycipitaceae and order Tremellales. These data emphasize the complexity of fungal diversity of unknown ecological function within a leaf that might influence plant disease epidemics or lead to the development of species for biocontrol of fungal disease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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September 22, 2019

Laboratory colonization stabilizes the naturally dynamic microbiome composition of field collected Dermacentor andersoni ticks.

Nearly a quarter of emerging infectious diseases identified in the last century are arthropod-borne. Although ticks and insects can carry pathogenic microorganisms, non-pathogenic microbes make up the majority of their microbial communities. The majority of tick microbiome research has had a focus on discovery and description; very few studies have analyzed the ecological context and functional responses of the bacterial microbiome of ticks. The goal of this analysis was to characterize the stability of the bacterial microbiome of Dermacentor andersoni ticks between generations and two populations within a species.The bacterial microbiome of D. andersoni midguts and salivary glands was analyzed from populations collected at two different ecologically distinct sites by comparing field (F1) and lab-reared populations (F1-F3) over three generations. The microbiome composition of pooled and individual samples was analyzed by sequencing nearly full-length 16S rRNA gene amplicons using a Pacific Biosciences CCS platform that allows identification of bacteria to the species level.In this study, we found that the D. andersoni microbiome was distinct in different geographic populations and was tissue specific, differing between the midgut and the salivary gland, over multiple generations. Additionally, our study showed that the microbiomes of laboratory-reared populations were not necessarily representative of their respective field populations. Furthermore, we demonstrated that the microbiome of a few individual ticks does not represent the microbiome composition at the population level.We demonstrated that the bacterial microbiome of D. andersoni was complex over three generations and specific to tick tissue (midgut vs. salivary glands) as well as geographic location (Burns, Oregon vs. Lake Como, Montana vs. laboratory setting). These results provide evidence that habitat of the tick population is a vital component of the complexity of the bacterial microbiome of ticks, and that the microbiome of lab colonies may not allow for comparative analyses with field populations. A broader understanding of microbiome variation will be required if we are to employ manipulation of the microbiome as a method for interfering with acquisition and transmission of tick-borne pathogens.

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September 22, 2019

Indoleacrylic acid produced by commensal Peptostreptococcus species suppresses inflammation.

Host factors in the intestine help select for bacteria that promote health. Certain commensals can utilize mucins as an energy source, thus promoting their colonization. However, health conditions such as inflammatory bowel disease (IBD) are associated with a reduced mucus layer, potentially leading to dysbiosis associated with this disease. We characterize the capability of commensal species to cleave and transport mucin-associated monosaccharides and identify several Clostridiales members that utilize intestinal mucins. One such mucin utilizer, Peptostreptococcus russellii, reduces susceptibility to epithelial injury in mice. Several Peptostreptococcus species contain a gene cluster enabling production of the tryptophan metabolite indoleacrylic acid (IA), which promotes intestinal epithelial barrier function and mitigates inflammatory responses. Furthermore, metagenomic analysis of human stool samples reveals that the genetic capability of microbes to utilize mucins and metabolize tryptophan is diminished in IBD patients. Our data suggest that stimulating IA production could promote anti-inflammatory responses and have therapeutic benefits. Copyright © 2017 Elsevier Inc. All rights reserved.

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September 22, 2019

Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection.

Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions. A total of 336 organisms were present in available rumen metagenomic data sets, and 134 were present in human gut microbiome data sets. Comparison with the human microbiome revealed rumen-specific enrichment for genes encoding de novo synthesis of vitamin B12, ongoing evolution by gene loss and potential vertical inheritance of the rumen microbiome based on underrepresentation of markers of environmental stress. We estimate that our Hungate genome resource represents ~75% of the genus-level bacterial and archaeal taxa present in the rumen.

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September 22, 2019

Comparative genome and transcriptome analysis reveals distinctive surface characteristics and unique physiological potentials of Pseudomonas aeruginosa ATCC 27853.

Pseudomonas aeruginosa ATCC 27853 was isolated from a hospital blood specimen in 1971 and has been widely used as a model strain to survey antibiotics susceptibilities, biofilm development, and metabolic activities of Pseudomonas spp.. Although four draft genomes of P. aeruginosa ATCC 27853 have been sequenced, the complete genome of this strain is still lacking, hindering a comprehensive understanding of its physiology and functional genome.Here we sequenced and assembled the complete genome of P. aeruginosa ATCC 27853 using the Pacific Biosciences SMRT (PacBio) technology and Illumina sequencing platform. We found that accessory genes of ATCC 27853 including prophages and genomic islands (GIs) mainly contribute to the difference between P. aeruginosa ATCC 27853 and other P. aeruginosa strains. Seven prophages were identified within the genome of P. aeruginosa ATCC 27853. Of the predicted 25 GIs, three contain genes that encode monoxoygenases, dioxygenases and hydrolases that could be involved in the metabolism of aromatic compounds. Surveying virulence-related genes revealed that a series of genes that encode the B-band O-antigen of LPS are lacking in ATCC 27853. Distinctive SNPs in genes of cellular adhesion proteins such as type IV pili and flagella biosynthesis were also observed in this strain. Colony morphology analysis confirmed an enhanced biofilm formation capability of ATCC 27853 on solid agar surface compared to Pseudomonas aeruginosa PAO1. We then performed transcriptome analysis of ATCC 27853 and PAO1 using RNA-seq and compared the expression of orthologous genes to understand the functional genome and the genomic details underlying the distinctive colony morphogenesis. These analyses revealed an increased expression of genes involved in cellular adhesion and biofilm maturation such as type IV pili, exopolysaccharide and electron transport chain components in ATCC 27853 compared with PAO1. In addition, distinctive expression profiles of the virulence genes lecA, lasB, quorum sensing regulators LasI/R, and the type I, III and VI secretion systems were observed in the two strains.The complete genome sequence of P. aeruginosa ATCC 27853 reveals the comprehensive genetic background of the strain, and provides genetic basis for several interesting findings about the functions of surface associated proteins, prophages, and genomic islands. Comparative transcriptome analysis of P. aeruginosa ATCC 27853 and PAO1 revealed several classes of differentially expressed genes in the two strains, underlying the genetic and molecular details of several known and yet to be explored morphological and physiological potentials of P. aeruginosa ATCC 27853.

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September 22, 2019

Targeted combinatorial alternative splicing generates brain region-specific repertoires of neurexins.

Molecular diversity of surface receptors has been hypothesized to provide a mechanism for selective synaptic connectivity. Neurexins are highly diversified receptors that drive the morphological and functional differentiation of synapses. Using a single cDNA sequencing approach, we detected 1,364 unique neurexin-a and 37 neurexin-ß mRNAs produced by alternative splicing of neurexin pre-mRNAs. This molecular diversity results from near-exhaustive combinatorial use of alternative splice insertions in Nrxn1a and Nrxn2a. By contrast, Nrxn3a exhibits several highly stereotyped exon selections that incorporate novel elements for posttranscriptional regulation of a subset of transcripts. Complexity of Nrxn1a repertoires correlates with the cellular complexity of neuronal tissues, and a specific subset of isoforms is enriched in a purified cell type. Our analysis defines the molecular diversity of a critical synaptic receptor and provides evidence that neurexin diversity is linked to cellular diversity in the nervous system. Copyright © 2014 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

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

The human microbiome and understanding the 16S rRNA gene in translational nursing science.

As more is understood regarding the human microbiome, it is increasingly important for nurse scientists and healthcare practitioners to analyze these microbial communities and their role in health and disease. 16S rRNA sequencing is a key methodology in identifying these bacterial populations that has recently transitioned from use primarily in research to having increased utility in clinical settings.The objectives of this review are to (a) describe 16S rRNA sequencing and its role in answering research questions important to nursing science; (b) provide an overview of the oral, lung, and gut microbiomes and relevant research; and (c) identify future implications for microbiome research and 16S sequencing in translational nursing science.Sequencing using the 16S rRNA gene has revolutionized research and allowed scientists to easily and reliably characterize complex bacterial communities. This type of research has recently entered the clinical setting, one of the best examples involving the use of 16S sequencing to identify resistant pathogens, thereby improving the accuracy of bacterial identification in infection control. Clinical microbiota research and related requisite methods are of particular relevance to nurse scientists-individuals uniquely positioned to utilize these techniques in future studies in clinical settings.

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