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1.
Cureus ; 16(2): e54233, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38496085

ABSTRACT

Surgery is a common and often necessary treatment option for a wide range of medical conditions, with an estimated 40 to 50 million surgeries performed in the US alone each year. While the various types of surgeries performed may be effective in treating or managing different conditions, the post-operative period can be challenging for patients. Osteopathic manipulative treatment (OMT) is a hands-on approach to medical care that seeks to restore balance and harmony to the body from the lens of an interconnected mind, body, and spirit. Given the potential for adverse events in patients following surgical treatments, OMT may be a viable adjunct post-operatively to enhance patient care and recovery. The purpose of this scoping review is to evaluate the state of current research examining the effectiveness of OMT in improving outcomes in post-operative patients. Three hundred articles were collected; 53 duplicates were removed. Eleven independent reviewers evaluated all 247 articles. Thirty articles were identified, including nine in general surgery, six in cardiothoracic surgery, five in orthopedic surgery, four in spinal surgery, three in neurosurgery, and three others (otolaryngology, oral/maxillofacial, and gynecologic surgery). Post-operative patients were treated with various OMT techniques with myofascial release and muscle energy being some of the most common treatments utilized in all surgical fields. Many studies demonstrated the benefits of OMT usage including significant pain relief, improved and earlier bowel function, and decreased lengths of hospital stay. This study demonstrates how OMT can be effective in reducing post-operative pain, reducing the incidence of post-operative ileus, and shortening the length of stay. Further research into the utilization of OMT in post-operative patients should be considered a potential adjunct to surgical intervention, especially in vulnerable patient populations.

2.
Qual Health Res ; 33(8-9): 801-810, 2023 07.
Article in English | MEDLINE | ID: mdl-37328281

ABSTRACT

Despite the senses being a valuable source of knowledge, little research has explored the sensory process of medical experiences. This narrative ethnographic study investigated how the senses shaped parents' experiences of waiting for their child to receive a solid organ, stem cell, or bone marrow transplant. Six parents from four different families primarily participated in sensory interviews as well as observations that explored the question: How do parents experience waiting using the five senses? Our narrative analysis suggested that parents' bodies stored sense memories, and they re-experienced stories of waiting through the senses and 'felt realities'. In addition, the senses transported families back to the emotional experience of waiting, which highlighted the longevity of waiting after receiving a transplant. We discuss how the senses provide important information about the body, waiting experiences, and the environmental contexts that mediate waiting. Findings contribute to theoretical and methodological work exploring how bodies are implicated in producing narratives.


Subject(s)
Narration , Parents , Humans , Child , Parents/psychology , Emotions , Anthropology, Cultural , Qualitative Research
3.
Microorganisms ; 12(1)2023 Dec 29.
Article in English | MEDLINE | ID: mdl-38257897

ABSTRACT

In an attempt to isolate new probiotic bacteria, two Gram-variable, spore-forming, rod-shaped aerobic bacteria designated as strain A4 and A15 were isolated from the feces of Canada geese (Branta canadensis). Strain A4 was able to grow in high salt levels and exhibited lipase activity, while A15 did not propagate under these conditions. Both were positive for starch hydrolysis, and they inhibited the growth of Staphylococcus aureus. The strains of the 16S rRNA sequence shared only 94% similarity to previously identified Sporosarcina spp. The ANI (78.08%) and AAI (82.35%) between the two strains were less than the species threshold. Searches for the most similar genomes using the Mash/Minhash algorithm showed the nearest genome to strain A4 and A15 as Sporosarcina sp. P13 (distance of 21%) and S. newyorkensis (distance of 17%), respectively. Sporosarcina spp. strains A4 and A15 contain urease genes, and a fibronectin-binding protein gene indicates that these bacteria may bind to eukaryotic cells in host gastrointestinal tracts. Phenotypic and phylogenetic data, along with low dDDH, ANI, and AAI values for strains A4 and A15, indicate these bacteria are two novel isolates of the Sporosarcina genus: Sporosarcina sp. A4 sp. nov., type strain as Sporosarcina cascadiensis and Sporosarcina sp. A15 sp. nov., type strain Sporosarcina obsidiansis.

4.
Front Psychol ; 12: 699088, 2021.
Article in English | MEDLINE | ID: mdl-34335417

ABSTRACT

The socio-economic benefits of interventions to prevent stress and related mental health problems are enormous. In the labor market, it is becoming desirable to keep employees for as long as possible. Since aging implies additional stressors such as increased risk of illness, and added pressure by professional tasks such as transferring knowledge, or learning new technologies, it is of particular relevance to offer stress-reduction to pre-retirement employees. Here, we report the effects of an eight-week Mindfulness-Based Stress Reduction (MBSR) intervention on mental well-being in 60-65-year-old work-active Danish employees, compared to a waiting-list control group. We observed improvements in resilience (Brief Resilience Scale) and mental well-being (WHO-5) not only at the end of the intervention, but also at the 12-month follow-up measurement that was preceded by monthly booster sessions. Interestingly, whereas well-being usually refers to experiences in the past weeks or months, we observed increasing Comfort in the MBSR-intervention group during a 5-minute eyes-closed rest session suggesting that this therapeutic effect of MBSR is measurable in how we feel even during short periods of time. We argue that MBSR is a cost-effective intervention suited for pre-retirement employees to cultivate resilience to prevent stress, feel more comfortable with themselves, maintain a healthy work-life in the last years before retirement, and, potentially, stay in their work-life a few more years than originally planned.

5.
Microbiol Resour Announc ; 9(10)2020 Mar 05.
Article in English | MEDLINE | ID: mdl-32139578

ABSTRACT

Here, we present the draft genome sequences of two Paenibacillus strains, An7 and USDA918EY, isolated from goose feces (Bend, OR, USA) and chicken ceca (Pomona, CA, USA), respectively. These data may assist with analyses of microorganisms associated with free-ranging and commercial avian species.

6.
G3 (Bethesda) ; 9(10): 3453-3465, 2019 10 07.
Article in English | MEDLINE | ID: mdl-31444295

ABSTRACT

The Neurospora crassa nuclear aod-1 gene encodes an alternative oxidase that functions in mitochondria. The enzyme provides a branch from the standard electron transport chain by transferring electrons directly from ubiquinol to oxygen. In standard laboratory strains, aod-1 is transcribed at very low levels under normal growth conditions. However, if the standard electron transport chain is disrupted, aod-1 mRNA expression is induced and the AOD1 protein is produced. We previously identified a strain of N. crassa, that produces high levels of aod-1 transcript under non-inducing conditions. Here we have crossed this strain to a standard lab strain and determined the genomic sequences of the parents and several progeny. Analysis of the sequence data and the levels of aod-1 mRNA in uninduced cultures revealed that a frameshift mutation in the flbA gene results in the high uninduced expression of aod-1 The flbA gene encodes a regulator of G protein signaling that decreases the activity of the Gα subunit of heterotrimeric G proteins. Our data suggest that strains with a functional flbA gene prevent uninduced expression of aod-1 by inactivating a G protein signaling pathway, and that this pathway is activated in cells grown under conditions that induce aod-1 Induced cells with a deletion of the gene encoding the Gα protein still have a partial increase in aod-1 mRNA levels, suggesting a second pathway for inducing transcription of the gene in N. crassa We also present evidence that a translational control mechanism prevents production of AOD1 protein in uninduced cultures.


Subject(s)
GTP-Binding Proteins/genetics , Gene Expression Regulation, Fungal , Mitochondrial Proteins/biosynthesis , Neurospora crassa/genetics , Neurospora crassa/metabolism , Oxidoreductases/biosynthesis , Plant Proteins/biosynthesis , Mutation , RNA, Messenger/genetics , RNA, Messenger/metabolism
7.
Fungal Genet Biol ; 132: 103256, 2019 11.
Article in English | MEDLINE | ID: mdl-31344458

ABSTRACT

Many secondary metabolites are produced by biosynthetic gene clusters (BGCs) that are repressed during standard growth conditions, which complicates the discovery of novel bioactive compounds. In the genus Fusarium, many BGCs reside in chromatin enriched for trimethylated histone 3 lysine 27 (H3K27me3), a modification correlated with transcriptional gene silencing. Here we report on our progress in assigning metabolites to genes by using a strain lacking the H3K27 methyltransferase, Kmt6. To guide isolation efforts, we coupled genetics to multivariate analysis of liquid chromatography-mass spectrometry (LCMS) data from both wild type and kmt6, which allowed identification of compounds previously unknown from F. graminearum. We found low molecular weight, amino acid-derived metabolites (N-ethyl anthranilic acid, N-phenethylacetamide, N-acetyltryptamine). We identified one new compound, protofusarin, as derived from fusarin biosynthesis. Similarly, we isolated large amounts of fusaristatin A, gibepyrone A, and fusarpyrones A and B, simply by using the kmt6 mutant, instead of having to optimize growth media. To increase the abundance of metabolites underrepresented in wild type, we generated kmt6 fus1 double mutants and discovered tricinolone and tricinolonoic acid, two new sesquiterpenes belonging to the tricindiol class. Our approach allows rapid visualization and analyses of the genetically induced changes in metabolite production, and discovery of new molecules by a combination of chemical and genetic dereplication. Of 22 fungal metabolites identified here, 10 compounds had not been reported from F. graminearum before. We show that activating silent metabolic pathways by mutation of a repressive chromatin modification enzyme can result in the discovery of new chemistry even in a well-studied organism, and helps to connect new or known small molecules to the BGCs responsible for their production.


Subject(s)
Fusarium/genetics , Fusarium/metabolism , Histone Code/genetics , Metabolomics , Secondary Metabolism/genetics , Biosynthetic Pathways/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Histone Methyltransferases/genetics , Mutation , Protein Processing, Post-Translational
8.
J Insect Physiol ; 117: 103891, 2019.
Article in English | MEDLINE | ID: mdl-31176625

ABSTRACT

The honey bee, Apis mellifera L., is a major pollinator insect that lacks novel "selenoprotein genes", rendering it susceptible to elevated levels of Selenium (Se) occurring naturally in the environment. We investigated the effects of two inorganic forms of Se on biological traits, oxidative stress, and gene regulation. Using bioassay arenas in the laboratory, one-day old sister bees were fed ad libitum 4 different concentrations of selenate and selenite, two common inorganic forms of Se. The transcription levels of 4 honey bee antioxidant genes were evaluated, and three putative selenoprotein-like genes (SELENOT, SELENOK, SELENOF) were characterized as well as Sbp2, a Selenium binding protein required for the translation of selenoproteins mRNA. Oxidative stress and Se residues were subsequently quantified in honey bee bodies throughout the experiment. Se induced higher oxidative stress in treated honey bees leading to a significantly elevated protein carbonyl content, particularly at the highest studied concentrations. Early upregulations of Spb2 and MsrA were identified at day 2 of the treatment while all genes except SELENOT were upregulated substantially at day 8 to alleviate the Se-induced oxidative stress levels. We determined that doses between 60 and 600 mg.Se.L-1 were acutely toxic to bees (<48 h) while doses between 0.6 and 6 mg.Se.L-1 led to much lower mortality (7-16)%. Furthermore, when fed ad libitum, Se residue data indicated that bees tolerated accumulation up to 0.12 µg Se bee-1 for at least 8 days with a Se LC50 of ∼6 mg/L, a field realistic concentration found in pollen of certain plants in a high Se soil environment.


Subject(s)
Bees/drug effects , Oxidative Stress/drug effects , Selenium Compounds/toxicity , Selenium/toxicity , Selenoproteins/genetics , Animals , Bees/genetics , Bees/metabolism , Gene Expression Regulation , Genes, Insect , Selenium/metabolism , Selenium Compounds/metabolism
9.
PLoS Genet ; 15(4): e1008093, 2019 04.
Article in English | MEDLINE | ID: mdl-31009462

ABSTRACT

Chromosome and genome stability are important for normal cell function as instability often correlates with disease and dysfunction of DNA repair mechanisms. Many organisms maintain supernumerary or accessory chromosomes that deviate from standard chromosomes. The pathogenic fungus Zymoseptoria tritici has as many as eight accessory chromosomes, which are highly unstable during meiosis and mitosis, transcriptionally repressed, show enrichment of repetitive elements, and enrichment with heterochromatic histone methylation marks, e.g., trimethylation of H3 lysine 9 or lysine 27 (H3K9me3, H3K27me3). To elucidate the role of heterochromatin on genome stability in Z. tritici, we deleted the genes encoding the methyltransferases responsible for H3K9me3 and H3K27me3, kmt1 and kmt6, respectively, and generated a double mutant. We combined experimental evolution and genomic analyses to determine the impact of these deletions on chromosome and genome stability, both in vitro and in planta. We used whole genome sequencing, ChIP-seq, and RNA-seq to compare changes in genome and chromatin structure, and differences in gene expression between mutant and wildtype strains. Analyses of genome and ChIP-seq data in H3K9me3-deficient strains revealed dramatic chromatin reorganization, where H3K27me3 is mostly relocalized into regions that are enriched with H3K9me3 in wild type. Many genome rearrangements and formation of new chromosomes were found in the absence of H3K9me3, accompanied by activation of transposable elements. In stark contrast, loss of H3K27me3 actually increased the stability of accessory chromosomes under normal growth conditions in vitro, even without large scale changes in gene activity. We conclude that H3K9me3 is important for the maintenance of genome stability because it disallows H3K27me3 in regions considered constitutive heterochromatin. In this system, H3K27me3 reduces the overall stability of accessory chromosomes, generating a "metastable" state for these quasi-essential regions of the genome.


Subject(s)
Genomic Instability , Histones/metabolism , Lysine/metabolism , Ascomycota/genetics , Ascomycota/metabolism , Chromosomes, Fungal , Gene Deletion , Heterochromatin/genetics , Heterochromatin/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histones/chemistry , Methylation , Repetitive Sequences, Nucleic Acid , Transcriptional Activation
11.
PLoS Genet ; 14(7): e1007511, 2018 07.
Article in English | MEDLINE | ID: mdl-30044771

ABSTRACT

The NF-κB-like velvet domain protein VosA (viability of spores) binds to more than 1,500 promoter sequences in the filamentous fungus Aspergillus nidulans. VosA inhibits premature induction of the developmental activator gene brlA, which promotes asexual spore formation in response to environmental cues as light. VosA represses a novel genetic network controlled by the sclB gene. SclB function is antagonistic to VosA, because it induces the expression of early activator genes of asexual differentiation as flbC and flbD as well as brlA. The SclB controlled network promotes asexual development and spore viability, but is independent of the fungal light control. SclB interactions with the RcoA transcriptional repressor subunit suggest additional inhibitory functions on transcription. SclB links asexual spore formation to the synthesis of secondary metabolites including emericellamides, austinol as well as dehydroaustinol and activates the oxidative stress response of the fungus. The fungal VosA-SclB regulatory system of transcription includes a VosA control of the sclB promoter, common and opposite VosA and SclB control functions of fungal development and several additional regulatory genes. The relationship between VosA and SclB illustrates the presence of a convoluted surveillance apparatus of transcriptional control, which is required for accurate fungal development and the linkage to the appropriate secondary metabolism.


Subject(s)
Aspergillus nidulans/physiology , Fungal Proteins/genetics , Oxidative Stress/genetics , Reproduction, Asexual/genetics , Secondary Metabolism/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal/physiology , Gene Regulatory Networks/physiology , Genes, Fungal/genetics , Promoter Regions, Genetic/genetics , Protein Domains/physiology , Spores, Fungal/genetics , Spores, Fungal/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Zinc Fingers/physiology
12.
Front Cardiovasc Med ; 4: 35, 2017.
Article in English | MEDLINE | ID: mdl-28612008

ABSTRACT

BACKGROUND: The instantaneous wave-free ratio (iFR) is a novel method to assess the ischemic potential of coronary artery stenoses. Clinical trial data have shown that iFR has acceptable diagnostic agreement with fractional flow reserve (FFR), the reference standard for the functional assessment of coronary stenoses. This study compares iFR measurements with FFR measurements in a real world, single-center setting. METHODS AND RESULTS: Instantaneous wave-free ratio and FFR were measured in 50 coronary artery lesions in 42 patients, with FFR ≤ 0.8 classified as functionally significant. An iFR-only technique, using a treatment cut-off value, iFR ≤ 0.89, provided a classification agreement of 84% with FFR ≤ 0.80. Use of a hybrid iFR-FFR technique, incorporating FFR measurement for lesions within the iFR gray zone of 0.86-0.93, would improve classification agreement with FFR to 94%, with diagnosis achieved without the need for hyperemia in 57% patients. CONCLUSION: This study in a real-world setting demonstrated good classification agreement between iFR and FFR. Use of a hybrid iFR-FFR technique would achieve high diagnostic accuracy while minimizing adenosine use, compared with routine FFR.

13.
mBio ; 8(3)2017 06 27.
Article in English | MEDLINE | ID: mdl-28655822

ABSTRACT

Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700-nucleotide (nt) 5' leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5' region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5' conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitroIn vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression.IMPORTANCE There is a deepening and widening appreciation of the diverse roles of translation in controlling gene expression. A central fungal transcription factor, the best-studied example of which is Saccharomyces cerevisiae GCN4, is crucial for the response to amino acid limitation. Two upstream open reading frames (uORFs) in the GCN4 mRNA are critical for controlling GCN4 synthesis. We observed that two uORFs in the corresponding Neurospora crassa cpc-1 mRNA appear functionally analogous to the GCN4 uORFs. We also discovered that, surprisingly, unlike GCN4, the CPC1 coding sequence extends far upstream from the presumed AUG start codon with no other in-frame AUG codons. Similar extensions were seen in homologs from many filamentous fungi. We observed that multiple non-AUG near-cognate codons (NCCs) in this extended reading frame, some conserved, initiated translation to produce longer forms of CPC1, underscoring the significance of noncanonical initiation in controlling gene expression.


Subject(s)
Codon , Gene Expression Regulation, Fungal , Neurospora crassa/genetics , Peptide Chain Initiation, Translational , Ascomycota/genetics , Basidiomycota/genetics , Gene Fusion , Open Reading Frames , Protein Biosynthesis , Saccharomyces cerevisiae/genetics
14.
G3 (Bethesda) ; 7(2): 449-466, 2017 02 09.
Article in English | MEDLINE | ID: mdl-27986792

ABSTRACT

In Neurospora crassa, blocking the function of the standard mitochondrial electron transport chain results in the induction of an alternative oxidase (AOX). AOX transfers electrons directly from ubiquinol to molecular oxygen. AOX serves as a model of retrograde regulation since it is encoded by a nuclear gene that is regulated in response to signals from mitochondria. The N. crassa transcription factors AOD2 and AOD5 are necessary for the expression of the AOX gene. To gain insight into the mechanism by which these factors function, and to determine if they have roles in the expression of additional genes in N. crassa, we constructed strains expressing only tagged versions of the proteins. Cell fractionation experiments showed that both proteins are localized to the nucleus under both AOX inducing and noninducing conditions. Furthermore, chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) analysis revealed that the proteins are bound to the promoter region of the AOX gene under both conditions. ChIP-seq also showed that the transcription factors bind to the upstream regions of a number of genes that are involved in energy production and metabolism. Dependence on AOD2 and AOD5 for the expression of several of these genes was verified by quantitative PCR. The majority of ChIP-seq peaks observed were enriched for both AOD2 and AOD5. However, we also observed occasional sites where one factor appeared to bind preferentially. The most striking of these was a conserved sequence that bound large amounts of AOD2 but little AOD5. This sequence was found within a 310 bp repeat unit that occurs at several locations in the genome.


Subject(s)
Energy Metabolism/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Fungal/genetics , Mitochondrial Proteins/genetics , Neurospora crassa/genetics , Oxidoreductases/genetics , Plant Proteins/genetics , Cell Nucleus/genetics , Genome, Fungal , Mitochondria/metabolism , Mutation , Neurospora crassa/metabolism , Transcription Factors/genetics
15.
G3 (Bethesda) ; 7(1): 129-142, 2017 01 05.
Article in English | MEDLINE | ID: mdl-27856696

ABSTRACT

Light and the circadian clock have a profound effect on the biology of organisms through the regulation of large sets of genes. Toward understanding how light and the circadian clock regulate gene expression, we used genome-wide approaches to identify the direct and indirect targets of the light-responsive and clock-controlled transcription factor ADV-1 in Neurospora crassa A large proportion of ADV-1 targets were found to be light- and/or clock-controlled, and enriched for genes involved in development, metabolism, cell growth, and cell fusion. We show that ADV-1 is necessary for transducing light and/or temporal information to its immediate downstream targets, including controlling rhythms in genes critical to somatic cell fusion. However, while ADV-1 targets are altered in predictable ways in Δadv-1 cells in response to light, this is not always the case for rhythmic target gene expression. These data suggest that a complex regulatory network downstream of ADV-1 functions to generate distinct temporal dynamics of target gene expression relative to the central clock mechanism.


Subject(s)
Circadian Clocks/genetics , Gene Regulatory Networks/genetics , Neurospora crassa/genetics , Transcription Factors/genetics , Circadian Clocks/physiology , Circadian Rhythm/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Genome, Fungal , Light , Neurospora crassa/physiology
16.
BMC Res Notes ; 8: 733, 2015 Nov 30.
Article in English | MEDLINE | ID: mdl-26621351

ABSTRACT

BACKGROUND: Given the widespread use of smartphone pedometer applications and the relatively limited number of published validity tests, this study examined the validity of three popular commercial smartphone pedometer applications (i.e., Accupedo, Moves, and Runtastic Pedometer). PARTICIPANTS: Convenience samples of males and females were recruited for laboratory tests [n = 11; mean: aged 24.18 years (±3.06)] and a free-living test [n = 18; mean: aged 28.78 years (±9.52)]. METHODS: Five conditions were assessed: (a) 20-step test, (b) 40-step stair climbing, (c) treadmill walking and running at different speeds, (d) driving, and (e) 3-day free-living. The Yamax SW-200 pedometer and observed step counts were used as criterion measures. RESULTS: Analyses identified an unacceptable error percentage in all of the applications compared to the pedometer. CONCLUSIONS: Given the inaccuracy of these applications, caution is required in their promotion to the public for self-monitoring physical activity and in their use as tools for assessing physical activity in research trials.


Subject(s)
Actigraphy/instrumentation , Automobile Driving , Exercise Test/instrumentation , Running , Walking , Actigraphy/methods , Adult , Exercise Test/methods , Female , Humans , Male , Middle Aged , Reproducibility of Results , Smartphone , Young Adult
17.
Article in English | MEDLINE | ID: mdl-26430472

ABSTRACT

BACKGROUND: Supernumerary chromosomes have been found in many organisms. In fungi, these "accessory" or "dispensable" chromosomes are present at different frequencies in populations and are usually characterized by higher repetitive DNA content and lower gene density when compared to the core chromosomes. In the reference strain of the wheat pathogen, Zymoseptoria tritici, eight discrete accessory chromosomes have been found. So far, no functional role has been assigned to these chromosomes; however, they have existed as separate entities in the karyotypes of Zymoseptoria species over evolutionary time. In this study, we addressed what-if anything-distinguishes the chromatin of accessory chromosomes from core chromosomes. We used chromatin immunoprecipitation combined with high-throughput sequencing ("ChIP-seq") of DNA associated with the centromere-specific histone H3, CENP-A (CenH3), to identify centromeric DNA, and ChIP-seq with antibodies against dimethylated H3K4, trimethylated H3K9 and trimethylated H3K27 to determine the relative distribution and proportion of euchromatin, obligate and facultative heterochromatin, respectively. RESULTS: Centromeres of the eight accessory chromosomes have the same sequence composition and structure as centromeres of the 13 core chromosomes and they are of similar length. Unlike those of most other fungi, Z. tritici centromeres are not composed entirely of repetitive DNA; some centromeres contain only unique DNA sequences, and bona fide expressed genes are located in regions enriched with CenH3. By fluorescence microscopy, we showed that centromeres of Z. tritici do not cluster into a single chromocenter during interphase. We found dramatically higher enrichment of H3K9me3 and H3K27me3 on the accessory chromosomes, consistent with the twofold higher proportion of repetitive DNA and poorly transcribed genes. In contrast, no single histone modification tested here correlated with the distribution of centromeric nucleosomes. CONCLUSIONS: All centromeres are similar in length and composed of a mixture of unique and repeat DNA, and most contain actively transcribed genes. Centromeres, subtelomeric regions or telomere repeat length cannot account for the differences in transfer fidelity between core and accessory chromosomes, but accessory chromosomes are greatly enriched in nucleosomes with H3K27 trimethylation. Genes on accessory chromosomes appear to be silenced by trimethylation of H3K9 and H3K27.

18.
G3 (Bethesda) ; 5(10): 1949-60, 2015 Jun 23.
Article in English | MEDLINE | ID: mdl-26109355

ABSTRACT

Genome defense likely evolved to curtail the spread of transposable elements and invading viruses. A combination of effective defense mechanisms has been shown to limit colonization of the Neurospora crassa genome by transposable elements. A novel DNA transposon named Sly1-1 was discovered in the genome of the most widely used laboratory "wild-type" strain FGSC 2489 (OR74A). Meiotic silencing by unpaired DNA, also simply called meiotic silencing, prevents the expression of regions of the genome that are unpaired during karyogamy. This mechanism is posttranscriptional and is proposed to involve the production of small RNA, so-called masiRNAs, by proteins homologous to those involved in RNA interference-silencing pathways in animals, fungi, and plants. Here, we demonstrate production of small RNAs when Sly1-1 was unpaired in a cross between two wild-type strains. These small RNAs are dependent on SAD-1, an RNA-dependent RNA polymerase necessary for meiotic silencing. We present the first case of endogenously produced masiRNA from a novel N. crassa DNA transposable element.


Subject(s)
DNA Transposable Elements/genetics , Gene Silencing , Meiosis/genetics , RNA, Small Untranslated/genetics , Amino Acid Sequence , Chromosome Mapping , Genetic Loci , Genome, Fungal , Molecular Sequence Data , Multigene Family , Neurospora crassa/classification , Neurospora crassa/genetics , Phylogeny , Sequence Alignment
19.
Proc Natl Acad Sci U S A ; 111(48): 16995-7002, 2014 Dec 02.
Article in English | MEDLINE | ID: mdl-25362047

ABSTRACT

Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation-based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter-luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level.


Subject(s)
Circadian Clocks , Gene Expression Regulation, Fungal , Neurospora crassa/genetics , Transcriptome/genetics , Energy Metabolism/genetics , Feedback, Physiological , Fungal Proteins/genetics , Fungal Proteins/metabolism , Genes, Fungal/genetics , High-Throughput Nucleotide Sequencing , Neurospora crassa/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, RNA , Signal Transduction/genetics
20.
PLoS One ; 9(9): e107672, 2014.
Article in English | MEDLINE | ID: mdl-25259845

ABSTRACT

Global change and its associated temperature increase has directly or indirectly changed the distributions of hosts and pathogens, and has affected host immunity, pathogen virulence and growth rates. This has resulted in increased disease in natural plant and animal populations worldwide, including scleractinian corals. While the effects of temperature increase on immunity and pathogen virulence have been clearly identified, their interaction, synergy and relative weight during pathogenesis remain poorly documented. We investigated these phenomena in the interaction between the coral Pocillopora damicornis and the bacterium Vibrio coralliilyticus, for which the infection process is temperature-dependent. We developed an experimental model that enabled unraveling the effects of thermal stress, and virulence vs. non-virulence of the bacterium. The physiological impacts of various treatments were quantified at the transcriptome level using a combination of RNA sequencing and targeted approaches. The results showed that thermal stress triggered a general weakening of the coral, making it more prone to infection, non-virulent bacterium induced an 'efficient' immune response, whereas virulent bacterium caused immuno-suppression in its host.


Subject(s)
Anthozoa/genetics , Anthozoa/immunology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Stress, Physiological , Temperature , Transcriptome , Vibrio , Animals , Anthozoa/microbiology , Cluster Analysis , Computational Biology , Gene Expression Profiling , Immunity, Innate/genetics , Indonesia , Reproducibility of Results
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