Sequence-dependent separation
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Allodiploid hybrid species, Aspergillus latus, belonging to section Nidulantes, is a hybrid of A. spinulosporus and an unknown species closely related to A. quadrilineatus and A. sublatus. This hybrid has often been misidentified as the species in section Nidulantes, such as A. nidulans, A. spinulosporus, A. sublatus, or other cryptic species. Aspergillus latus has not been reported in Japan as well as Asia so far. In this study, we screened 23 clinical strains identified as A. spinulosporus isolated in Japan from 2012 to 2023 and found seven A. latus strains. To characterize the A. latus strains, we conducted comprehensive phenotyping including morphological observation, whole genome sequences, and phylogenetic analysis based on calmodulin (CaM) gene. In addition, we conducted antifungal susceptibility testing for A. latus strains. As a result, the morphological characters of A. latus were more similar to those of A. spinulosporus compared to A. sublatus. However, the ascospore of A. latus differed from that of A. spinulosporus. Phylogenetic analysis revealed that different CaM alleles from the same isolate clustered separately with A. spinulosporus and A. sublatus, consistent with its hybrid origin. Furthermore, A. latus strains showed reduced susceptibility to caspofungin and amphotericin B compared to A. spinulosporus, while they were susceptible to azoles. Our results suggest that A. latus has been a causative pathogen of aspergillosis in Japan since 2013.
- MeSH
- antifungální látky farmakologie MeSH
- Aspergillus * genetika klasifikace izolace a purifikace účinky léků MeSH
- aspergilóza * mikrobiologie epidemiologie MeSH
- fylogeneze MeSH
- kalmodulin genetika MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- sekvenování celého genomu MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Japonsko MeSH
A novel Bartonella-like symbiont (BLS) of Tyrophagus putrescentiae was characterized. BLS formed a separate cluster from the Bartonella clade together with an ant symbiont. BLS was present in mite bodies (103 16S DNA copies/mite) and feces but was absent in eggs. This indicated the presence of the BLS in mite guts. The BLS showed a reduction in genome size (1.6 Mb) and indicates gene loss compared to Bartonella apis. The BLS can be interacted with its host by using host metabolic pathways (e.g., the histidine and arginine metabolic pathways) as well as by providing its own metabolic pathways (pantothenate and lipoic acid) to the host, suggesting the existence of a mutualistic association. Our experimental data further confirmed these potential mutualistic nutritional associations, as cultures of T. putrescentiae with low BLS abundance showed the strongest response after the addition of vitamins. Despite developing an arguably tight dependency on its host, the BLS has probably retained flagellar mobility, as evidenced by the 32 proteins enriched in KEGG pathways associated with flagellar assembly or chemotaxis (e.g., fliC, flgE, and flgK, as highly expressed genes). Some of these proteins probably also facilitate adhesion to host gut cells. The microcin C transporter was identified in the BLS, suggesting that microcin C may be used in competition with other gut bacteria. The 16S DNA sequence comparison indicated a mite clade of BLSs with a broad host range, including house dust and stored-product mites. Our phylogenomic analyses identified a unique lineage of arachnid specific BLSs in mites and scorpions.IMPORTANCEA Bartonella-like symbiont was found in an astigmatid mite of allergenic importance. We assembled the genome of the bacterium from metagenomes of different stored-product mite (T. putrescentiae) cultures. The bacterium provides pantothenate and lipoic acid to the mite host. The vitamin supply explains the changes in the relative abundance of BLSs in T. putrescentiae as the microbiome response to nutritional or pesticide stress, as observed previously. The phylogenomic analyses of available 16S DNA sequences originating from mite, scorpion, and insect samples identified a unique lineage of arachnid specific forming large Bartonella clade. BLSs associated with mites and a scorpion. The Bartonella clade included the previously described Ca. Tokpelaia symbionts of ants.
- MeSH
- Acaridae * mikrobiologie MeSH
- alergeny MeSH
- Bacteria MeSH
- Bartonella * genetika MeSH
- kyselina lipoová * MeSH
- roztoči * genetika MeSH
- symbióza MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Global impact of COVID-19 pandemic has heightened the urgency for efficient virus detection and identification of variants such as the Q57H mutation. Early and efficient detection of SARS-CoV-2 among densely populated developing countries is paramount objective. Although RT-PCR assays offer accuracy, however, dependence on expansive kits and availability of allied health resources pose an immense challenge for developing countries. In the current study, RT-LAMP based detection of SARS-Cov-2 with subsequent confirmation of Q57H variant through ARMS-PCR was performed. Among the 212 collected samples, 134 yielded positive results, while 78 tested negative using RT-LAMP. Oropharyngeal swabs of suspected individuals were collected and processed for viral RNA isolation. Isolated viral RNA was processed further by using either commercially available WarmStart Master Mix or our in house developed LAMP master mix separately. Subsequently, the end results of each specimen were evaluated by colorimetry. For LAMP assays, primers targeting three genes (ORF1ab, N and S) were designed using PrimerExplorer software. Interestingly, pooling of these three genes in single reaction tube increased sensitivity (95.5%) and specificity (93.5%) of LAMP assay. SARS-CoV-2 positive specimens were screened further for Q57H mutation using ARMS-PCR. Based on amplicon size variation, later confirmed by sequencing, our data showed 18.5% samples positive for Q57H mutation. Hence, these findings strongly advocate use of RT-LAMP-based assay for SARS-CoV-2 screening within suspected general population. Furthermore, ARMS-PCR also provides an efficient mean to detect prevalent mutations against SARS-Cov-2.
- MeSH
- COVID-19 * diagnóza MeSH
- diagnostické techniky molekulární metody MeSH
- lidé MeSH
- pandemie MeSH
- polymerázová řetězová reakce MeSH
- RNA virová genetika MeSH
- SARS-CoV-2 * genetika MeSH
- senzitivita a specificita MeSH
- testování na COVID-19 MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Preimplantation mouse embryo development involves temporal-spatial specification and segregation of three blastocyst cell lineages: trophectoderm, primitive endoderm and epiblast. Spatial separation of the outer-trophectoderm lineage from the two other inner-cell-mass (ICM) lineages starts with the 8- to 16-cell transition and concludes at the 32-cell stages. Accordingly, the ICM is derived from primary and secondary contributed cells; with debated relative EPI versus PrE potencies. We report generation of primary but not secondary ICM populations is highly dependent on temporal activation of mammalian target of Rapamycin (mTOR) during 8-cell stage M-phase entry, mediated via regulation of the 7-methylguanosine-cap (m7G-cap)-binding initiation complex (EIF4F) and linked to translation of mRNAs containing 5' UTR terminal oligopyrimidine (TOP-) sequence motifs, as knockdown of identified TOP-like motif transcripts impairs generation of primary ICM founders. However, mTOR inhibition-induced ICM cell number deficits in early blastocysts can be compensated by the late blastocyst stage, after inhibitor withdrawal; compensation likely initiated at the 32-cell stage when supernumerary outer cells exhibit molecular characteristics of inner cells. These data identify a novel mechanism specifically governing initial spatial segregation of mouse embryo blastomeres, that is distinct from those directing subsequent inner cell formation, contributing to germane segregation of late blastocyst lineages.
- MeSH
- blastocysta * MeSH
- buněčná diferenciace fyziologie MeSH
- buněčný rodokmen MeSH
- embryo savčí * MeSH
- mTORC1 MeSH
- myši MeSH
- savci MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytosine-rich DNA regions can form four-stranded structures based on hemi-protonated C.C+ pairs, called i-motifs (iMs). Using CD, UV absorption, NMR spectroscopy, and DSC calorimetry, we show that model (CnT3)3Cn (Cn) sequences adopt iM under neutral or slightly alkaline conditions for n > 3. However, the iMs are formed with long-lasting kinetics under these conditions and melt with significant hysteresis. Sequences with n > 6 melt in two or more separate steps, indicating the presence of different iM species, the proportion of which is dependent on temperature and incubation time. At ambient temperature, kinetically favored iMs of low stability are formed, most likely consisting of short C.C+ blocks. These species act as kinetic traps and prevent the assembly of thermodynamically favored, fully C.C+ paired iMs. A higher temperature is necessary to unfold the kinetic forms and enable their substitution by a slowly developing thermodynamic structure. This complicated kinetic partitioning process considerably slows down iM folding, making it much slower than the timeframes of biological reactions and, therefore, unlikely to have any biological relevance. Our data suggest kinetically driven iM species as more likely to be biologically relevant than thermodynamically most stable iM forms.
Circadian clocks are timing devices that rhythmically adjust organism's behavior, physiology, and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks rely on several interlocked transcription-translation feedback loops, where protein stability is the key part of the delay between transcription and the appearance of the mature proteins within the feedback loops. In bilaterian animals, including mammals and insects, the circadian clock depends on a homologous set of proteins. Despite mostly conserved clock components among the fruit fly Drosophila and mammals, several lineage-specific differences exist. Here we have systematically explored the evolution and sequence variability of insect DBT proteins and their vertebrate homologs casein kinase 1 delta (CKIδ) and epsilon (CKIε), dated the origin and separation of CKIδ from CKIε, and identified at least three additional independent duplications of the CKIδ/ε gene in Petromyzon, Danio, and Xenopus. We determined conserved regions in DBT specific to Diptera, and functionally tested a subset of those in D. melanogaster. Replacement of Lysine K224 with acidic residues strongly impacts the free-running period even in heterozygous flies, whereas homozygous mutants are not viable. K224D mutants have a temperature compensation defect with longer free-running periods at higher temperatures, which is exactly the opposite trend of what was reported for corresponding mammalian mutants. All DBTs of dipteran insects contain the NKRQK motif at positions 220-224. The occurrence of this motif perfectly correlates with the presence of BRIDE OF DOUBLETIME, BDBT, in Diptera. BDBT is a non-canonical FK506-binding protein that physically interacts with Drosophila DBT. The phylogeny of FK506-binding proteins suggests that BDBT is either absent or highly modified in non-dipteran insects. In addition to in silico analysis of DBT/CKIδ/ε evolution and diversity, we have identified four novel casein kinase 1 genes specific to the Drosophila genus.
- Publikační typ
- časopisecké články MeSH
The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.
- MeSH
- G-kvadruplexy * MeSH
- intergenová DNA chemie genetika MeSH
- lidé MeSH
- regulace genové exprese MeSH
- RNA virová MeSH
- RNA chemie genetika MeSH
- transport RNA MeSH
- virus Epsteinův-Barrové - jaderné antigeny chemie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Východiska: Prognóza pacientů s karcinomem kolorekta (colorectal cancer – CRC) závisí především na rozsahu onemocnění v době diagnózy, proto je brzký záchyt jedním z hlavních předpokladů úspěšné léčby. Současný výzkum ukazuje, že exozomální dlouhé nekódující RNA (lncRNA) jsou spojeny s rozvojem nádorových onemocnění. Jelikož jsou lncRNA často tkáňově specifické, jejich kvantifikace v exozomech se nabízí jako neinvazivní metoda pro včasnou detekci CRC. V naší práci jsme se zaměřili na optimalizaci protokolu pro analýzu exozomálních lncRNA z krevního séra pacientů s CRC jako potenciálních diagnostických biomarkerů. Materiál a metody: Exozomy byly izolovány pomocí gelové chromatografie ze 150 μl séra pacientů s CRC a zdravých dárců. Jejich kvalita a kvantita byla potvrzena elektronovou mikroskopií a analýzou dynamického rozptylu světla (dynamic light scattering – DLS) a proteinové markery byly detekovány metodou Western blot. Po izolaci RNA byly ze vzorků připraveny cDNA knihovny, které byly sekvenovány pomocí NextSeq 550. Výsledky: Úspěšně jsme izolovali exozomy a ověřili jsme jejich vlastnosti několika různými metodami. Knihovny byly připraveny ze všech vzorků i přes velmi nízký objem výchozího materiálu. Sekvenační data potvrzují přítomnost protein kódující (50 %) i nekódující RNA, kterou tvoří především lncRNA (28,2 %), pseudogeny (15,2 %) a další typy RNA (6,5 %). Výsledky dále ukázaly významně změněné hladiny některých lncRNA, na základě jejichž exprese bylo možné odlišit vzorky od pacientů s CRC od vzorků zdravých kontrol. Pomocí analýzy obohacení genové sady (gene set enrichment analysis – GSEA) jsme pozorovali významně obohacené třídy genů, které souvisejí s opravami DNA nebo regulací buněčného cyklu. Závěr: Naše pilotní data naznačují, že lncRNA představují významnou část RNA přítomné v exozomech a jejich rozdílné hladiny mají schopnost odlišit CRC pacienty od zdravých kontrol. Analýza obohacených genů zároveň prokázala významné zastoupení lncRNA podílejících se na regulaci buněčného cyklu a oprav DNA, což naznačuje jejich možné zapojení do procesů kancerogeneze. Výsledky je však třeba ověřit na větším souboru pacientů.
Background: The prognosis of patients with colorectal cancer (CRC) depends mainly on the extent of the disease at the time of diagnosis; therefore, early detection is one of the main prerequisites for successful treatment. Current research shows that exosomal long non-coding RNAs (lncRNAs) are associated with cancer development. As lncRNAs are often tissue specific, their quantification in exosomes is proposed as a non-invasive method for early detection of CRC. In this study, we aimed to optimize a protocol for analyzing exosomal lncRNAs from blood serum of CRC patients as potential diagnostic biomarkers. Material and methods: Exosomes were isolated by gel chromatography from 150 μl of serum of CRC patients and healthy donors. Their quality and quantity were confirmed by electron microscopy and dynamic light scattering (DLS) analysis; protein markers were detected by Western blot. After RNA isolation, cDNA libraries were prepared and sequenced using NextSeq 550. Results: We successfully isolated exosomes and verified them by several methods. Libraries were prepared from all samples despite very low volume of starting material. The sequencing data confirmed the presence of both protein-coding (50%) and non-coding RNAs, which consisted mainly of lncRNAs (28.2%), pseudogenes (15.2%) and other RNA types (6.5%). The results also showed significantly altered levels of some lncRNAs that could distinguish samples from CRC patients and healthy controls. Using gene set enrichment analysis (GSEA), we observed significantly enriched classes of genes related to DNA repair or cell cycle regulation. Conclusion: Our preliminary data suggest that lncRNAs represent a significant fraction of the RNA present in exosomes and that their distinct levels can separate CRC patients from healthy controls. The analysis of enriched genes also showed a significant representation of lncRNAs involved in cell cycle regulation and DNA repair, suggesting their possible involvement in cancerogenesis. However, the results need to be verified in a larger cohort of patients.
»: Fractures of the growing scapula account for about 0.1% of all pediatric fractures, with the majority occurring at an age of ≥10 years. »: Radiographic diagnosis requires a detailed knowledge of the ossification sequence of the growing scapula. Computed tomography examination is indicated for displaced fractures of the glenoid fossa, the scapular neck, and complex fractures; magnetic resonance imaging should be performed in cases of suspected injury to the physis and with stress fractures, depending on patient age and the potential need for general anesthesia during the scanning process. »: Separation of the base of the coracoid process is often associated with acromioclavicular dislocation. Clavicular fractures rarely occur in combination with injuries to the growing scapula. »: The majority of scapular fractures can be treated nonoperatively. Indications for surgery are displaced intra-articular fractures, scapular neck fractures with a displacement of >2 cm, coracoid base separation associated with acromioclavicular dislocation, and scapulothoracic dissociation. »: Displaced intra-articular fractures of the glenoid fossa should be followed after healing until skeletal maturity. Complications are rare and occur most frequently with scapulothoracic dissociation.
- MeSH
- dítě MeSH
- fraktury proximálního humeru * MeSH
- klíční kost diagnostické zobrazování zranění MeSH
- kloubní jamka lopatky * MeSH
- lidé MeSH
- lopatka chirurgie MeSH
- mladiství MeSH
- vnitřní fixace fraktury MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
OBJECTIVES: To test the performance of an oral cancer prognostic 13-gene signature for the prediction of survival of patients diagnosed with HPV-negative and p16-negative oral cavity cancer. MATERIALS AND METHODS: Diagnostic formalin-fixed paraffin-embedded oral cavity cancer tumor samples were obtained from the Fred Hutchinson Cancer Research Center/University of Washington, University of Calgary, University of Michigan, University of Utah, and seven ARCAGE study centers coordinated by the International Agency of Research on Cancer. RNA from 638 Human Papillomavirus (HPV)-negative and p16-negative samples was analyzed for the 13 genes using a NanoString assay. Ridge-penalized Cox regressions were applied to samples randomly split into discovery and validation sets to build models and evaluate the performance of the 13-gene signature in predicting 2-year oral cavity cancer-specific survival overall and separately for patients with early and late stage disease. RESULTS: Among AJCC stage I/II patients, including the 13-gene signature in the model resulted in substantial improvement in the prediction of 2-year oral cavity cancer-specific survival. For models containing age and sex with and without the 13-gene signature score, the areas under the Receiver Operating Characteristic Curve (AUC) and partial AUC were 0.700 vs. 0.537 (p < 0.001), and 0.046 vs. 0.018 (p < 0.001), respectively. Improvement in predicting prognosis for AJCC stage III/IV disease also was observed, but to a lesser extent. CONCLUSIONS: If confirmed using tumor samples from a larger number of early stage oral cavity cancer patients, the 13-gene signature may inform personalized treatment of early stage HPV-negative and p16-negative oral cavity cancer patients.
- MeSH
- analýza přežití MeSH
- dospělí MeSH
- fixace tkání MeSH
- inhibitor p16 cyklin-dependentní kinasy metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- lidský papilomavirus 16 izolace a purifikace MeSH
- mladý dospělý MeSH
- nádorové biomarkery genetika MeSH
- nádory úst genetika metabolismus patologie MeSH
- plocha pod křivkou MeSH
- sekvenční analýza RNA MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- spinocelulární karcinom genetika metabolismus patologie MeSH
- staging nádorů MeSH
- stanovení celkové genové exprese metody MeSH
- zalévání tkání do parafínu MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
