Bacteria employ small non-coding RNAs (sRNAs) to regulate gene expression. Ms1 is an sRNA that binds to the RNA polymerase (RNAP) core and affects the intracellular level of this essential enzyme. Ms1 is structurally related to 6S RNA that binds to a different form of RNAP, the holoenzyme bearing the primary sigma factor. 6S RNAs are widespread in the bacterial kingdom except for the industrially and medicinally important Actinobacteria. While Ms1 RNA was identified in Mycobacterium, it is not clear whether Ms1 RNA is present also in other Actinobacteria species. Here, using a computational search based on secondary structure similarities combined with a linguistic gene synteny approach, we identified Ms1 RNA in Streptomyces. In S. coelicolor, Ms1 RNA overlaps with the previously annotated scr3559 sRNA with an unknown function. We experimentally confirmed that Ms1 RNA/scr3559 associates with the RNAP core without the primary sigma factor HrdB in vivo. Subsequently, we applied the computational approach to other Actinobacteria and identified Ms1 RNA candidates in 824 Actinobacteria species, revealing Ms1 RNA as a widespread class of RNAP binding sRNAs, and demonstrating the ability of our multifactorial computational approach to identify weakly conserved sRNAs in evolutionarily distant genomes.
- Klíčová slova
- 6S RNA, Actinobacteria, Ms1 RNA, Mycobacterium, Streptomyces, gene synteny, sRNA,
- Publikační typ
- časopisecké články MeSH
Ms1 is a sRNA recently found in mycobacteria and several other actinobacterial species. Ms1 interacts with the RNA polymerase (RNAP) core devoid of sigma factors, which differs from 6S RNA that binds to RNAP holoenzymes containing the primary sigma factor. Here we show that Ms1 is the most abundant non-rRNA transcript in stationary phase in Mycobacterium smegmatis. The accumulation of Ms1 stems from its high-level synthesis combined with decreased degradation. We identify the Ms1 promoter, PMs1 , and cis-acting elements important for its activity. Furthermore, we demonstrate that PNPase (an RNase) contributes to the differential accumulation of Ms1 during growth. Then, by comparing the transcriptomes of wt and ΔMs1 strains from stationary phase, we reveal that Ms1 affects the intracellular level of RNAP. The absence of Ms1 results in decreased levels of the mRNAs encoding β and β' subunits of RNAP, which is also reflected at the protein level. Thus, the ΔMs1 strain has a smaller pool of RNAPs available when the transcriptional demand increases. This contributes to the inability of the ΔMs1 strain to rapidly react to environmental changes during outgrowth from stationary phase.
- MeSH
- bakteriální RNA metabolismus MeSH
- delece genu MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- malá nekódující RNA genetika metabolismus MeSH
- Mycobacterium smegmatis enzymologie genetika růst a vývoj metabolismus MeSH
- stanovení celkové genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální RNA MeSH
- DNA řízené RNA-polymerasy MeSH
- malá nekódující RNA MeSH
Small RNAs (sRNAs) are molecules essential for a number of regulatory processes in the bacterial cell. Here we characterize Ms1, a sRNA that is highly expressed in Mycobacterium smegmatis during stationary phase of growth. By glycerol gradient ultracentrifugation, RNA binding assay, and RNA co-immunoprecipitation, we show that Ms1 interacts with the RNA polymerase (RNAP) core that is free of the primary sigma factor (σA) or any other σ factor. This contrasts with the situation in most other species where it is 6S RNA that interacts with RNAP and this interaction requires the presence of σA. The difference in the interaction of the two types of sRNAs (Ms1 or 6S RNA) with RNAP possibly reflects the difference in the composition of the transcriptional machinery between mycobacteria and other species. Unlike Escherichia coli, stationary phase M. smegmatis cells contain relatively few RNAP molecules in complex with σA. Thus, Ms1 represents a novel type of small RNAs interacting with RNAP.
- MeSH
- bakteriální chromozomy MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- konformace nukleové kyseliny MeSH
- malá nekódující RNA chemie genetika metabolismus MeSH
- Mycobacterium smegmatis enzymologie genetika růst a vývoj MeSH
- Mycobacterium genetika MeSH
- sigma faktor metabolismus MeSH
- syntenie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA řízené RNA-polymerasy MeSH
- malá nekódující RNA MeSH
- sigma faktor MeSH
Histone post-translational modifications (hPTMs) are epigenetic marks that strongly affect numerous processes, including cell cycling and protein interactions. They have been studied by both antibody- and MS-based methods for years, but the analyses are still challenging, mainly because of the diversity of histones and their modifications arising from high contents of reactive amine groups in their amino acid sequences. Here, we introduce use of trimethylacetic anhydride (TMA) as a new reagent for efficient histone derivatization, which is a requirement for bottom-up proteomic hPTM analysis. TMA can derivatize unmodified amine groups of lysine residues and amine groups generated at peptide N-termini by trypsin digestion. The derivatization is facilitated by microwave irradiation, which also reduces incubation times to minutes. We demonstrate that histone derivatization with TMA reliably provides high yields of fully derivatized peptides and thus is an effective alternative to conventional methods. TMA afforded more than 98% and 99% labeling efficiencies for histones H4 and H3, respectively, thereby enabling accurate quantification of peptide forms. Trimethylacetylation substantially improves chromatographic separation of peptide forms, which is essential for direct quantification based on signals extracted from MS1 data. For this purpose, software widely applied by the proteomics community can be used without additional computational development. Thorough comparison with widely applied propionylation highlights the advantages of TMA-based histone derivatization for monitoring hPTMs in biological samples.
- Klíčová slova
- bottom–up proteomics, chemical derivatization, histone post-translational modifications, microwave irradiation, trimethylacetic anhydride,
- MeSH
- acetanhydridy chemie MeSH
- acetylace MeSH
- chromatografie kapalinová MeSH
- histony chemie MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- posttranslační úpravy proteinů MeSH
- tandemová hmotnostní spektrometrie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- acetanhydridy MeSH
- histony MeSH
Additive manufacturing (AM) is today in the main focus-and not only in commercial production. Products with complex geometry can be built using various AM techniques, which include laser sintering of metal powder. Although the technique has been known for a quite long time, the impact of the morphology of individual powder particles on the process has not yet been adequately documented. This article presents a detailed microscopic analysis of virgin and reused powder particles of MS1 maraging steel. The metallographic observation was performed using a scanning electron microscope (SEM). The particle size of the individual powder particles was measured in the SEM and the particle surface morphology and its change in the reused powder were observed. Individual particles were analyzed in detail using an SEM with a focused ion beam (FIB) milling capability. The powder particles were gradually cut off in thin layers so that their internal structure, chemical element distribution, possible internal defects, and shape could be monitored. Elemental distribution and phase distribution were analyzed using EDS and EBSD, respectively. Our findings lead to a better understanding and prediction of defects in additive-manufactured products. This could be helpful not just in the AM field, but in any metal powder-based processes, such as metal injection molding, powder metallurgy, spray deposition processes, and others.
- Klíčová slova
- EBSD, EDS, FIB, additive manufacturing, maraging steel,
- Publikační typ
- časopisecké články MeSH
Metal powders for additive manufacturing are expensive, and producing new ones from mined metals has a negative ecological impact. In this work, recycled and reused metal powders from MS1 steel for direct metal laser sintering (DMLS) 3D printing were evaluated in the laboratory. The powders were recycled by melting followed by gas atomizing. Virgin, recycled, and reused metal powders were evaluated using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), metallography analysis, microhardness measurements, particle size distribution (PSD), shape factor by digital image processing (DIP), and flowability testing. The results showed that the particle distribution was modified after recycling. Kurtosis analysis revealed a reduction from -0.64 for virgin powders to -1.29 for recycled powders. The results demonstrated a positive skewness, indicating that the recycled powder contained a greater proportion of smaller particles. The shape factor was also modified and changed from 1.57 for virgin powders to 1.28 for recycled powders. The microstructure also changed, and austenite was found in the recycled powders. The microhardness of recycled powder decreased by 39% compared to the virgin powder. Recycled powders did not flow, using two different funnels to evaluate their flowability. The flowability of used powder was reduced from 4.3 s to 2.9 s.
- Klíčová slova
- 3D printing, DMLS, MS1, additive manufacturing, metal powders, recycling,
- Publikační typ
- časopisecké články MeSH
The population genetics of the family Partitiviridae was studied within the European race of the conifer pathogen Gremmeniella abietina. One hundred sixty-two isolates were collected from different countries, including Canada, the Czech Republic, Finland, Italy, Montenegro, Serbia, Spain, Switzerland, Turkey and the United States. A unique species of G. abietina RNA virus-MS1 (GaRV-MS1) appears to occur indistinctly in G. abietina biotypes A and B, without a particular geographical distribution pattern. Forty-six isolates were shown to host GaRV-MS1 according to direct specific RT-PCR screening, and the virus was more common in biotype A than B. Phylogenetic analysis based on 46 partial coat protein (CP) cDNA sequences divided the GaRV-MS1 population into two closely related clades, while RNA-dependent RNA polymerase (RdRp) sequences revealed only one clade. The evolution of the virus appears to mainly occur through purifying selection but also through recombination. Recombination events were detected within alignments of the three complete CP and RdRp sequences of GaRV-MS1. This is the first time that recombination events have been directly identified in fungal partitiviruses and in G. abietina in particular. The results suggest that the population dynamics of GaRV-MS1 do not have a direct impact on the genetic structure of its host, G. abietina, though they might have had an innocuous ancestral relationship.
- Klíčová slova
- Biotype, Brunchorstia pinea, Conifers, Evolutio, Mycoviruses, Partitiviridae,
- MeSH
- Ascomycota izolace a purifikace virologie MeSH
- fylogeneze MeSH
- genotyp MeSH
- molekulární evoluce MeSH
- molekulární sekvence - údaje MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- rekombinace genetická MeSH
- RNA virová genetika MeSH
- RNA-viry klasifikace genetika izolace a purifikace MeSH
- sekvenční analýza DNA MeSH
- sekvenční homologie MeSH
- shluková analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
- Kanada MeSH
- Spojené státy americké MeSH
- Názvy látek
- RNA virová MeSH
Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.
- MeSH
- Bacillus subtilis genetika metabolismus MeSH
- bakteriální proteiny * metabolismus genetika MeSH
- bakteriální RNA * metabolismus genetika MeSH
- Corynebacterium glutamicum genetika metabolismus MeSH
- DNA řízené RNA-polymerasy * metabolismus genetika MeSH
- genetická transkripce MeSH
- konformace nukleové kyseliny MeSH
- Mycobacterium smegmatis genetika metabolismus enzymologie MeSH
- Mycobacterium tuberculosis genetika metabolismus MeSH
- nekódující RNA MeSH
- regulace genové exprese u bakterií MeSH
- sigma faktor * metabolismus genetika MeSH
- Streptomyces coelicolor genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 6S RNA MeSH Prohlížeč
- bakteriální proteiny * MeSH
- bakteriální RNA * MeSH
- DNA řízené RNA-polymerasy * MeSH
- nekódující RNA MeSH
- sigma faktor * MeSH
Dietary composition and metabolism of fatty acids (FA) influence insulin resistance, atherogenic dyslipidemia and other components of the metabolic syndrome (MS). It is known that patients with MS exhibit a heterogeneous phenotype; however, the relationships of individual FA to MS components have not yet been consistently studied. We examined the plasma phosphatidylcholine FA composition of 166 individuals (68F/98M) with MS and of 188 (87F/101M) controls. Cluster analysis of FA divided the groups into two clusters. In cluster 1, there were 65.7 % of MS patients and 37.8 % of controls, cluster 2 contained 34.3 % of patients and 62.2 % of controls (P<0.001). Those with MS within cluster 1 (MS1) differed from individuals with MS in cluster 2 (MS2) by concentrations of glucose (P<0.05), NEFA (P<0.001), HOMA-IR (P<0.05), and levels of conjugated dienes in LDL (P<0.05). The FA composition in MS1 group differed from MS2 by higher contents of palmitoleic (+30 %), gamma-linolenic (+22 %), dihomo-gamma-linolenic (+9 %) acids and by a lower content of linoleic acid (-25 %) (all P<0.01). These FA patterns are supposed to be connected with the progression and/or impaired biochemical measures of MS (lipolysis, oxidative stress, dysglycidemia, and insulin resistance).
- MeSH
- fosfatidylcholiny krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- mastné kyseliny krev MeSH
- metabolický syndrom krev klasifikace diagnóza MeSH
- senioři MeSH
- shluková analýza MeSH
- stravovací zvyklosti fyziologie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fosfatidylcholiny MeSH
- mastné kyseliny MeSH
Faecal samples were collected from 352 horses on 23 farms operating under six different management systems in the Czech Republic and Poland during 2011 and 2012. Farms were selected without previous knowledge of parasitological status. All faecal samples were screened for Cryptosporidium spp. presence using microscopy, following aniline-carbol-methyl violet staining and PCR analysis of the small-subunit (SSU) rRNA and the 60-kDa glycoprotein (gp60) genes. Cryptosporidium muris-positive samples were additionally genotyped at four minisatellite markers: MS1 (encoding a hypothetical protein), MS2 (encoding a 90-kDa heat shock protein), MS3 (encoding a hypothetical protein) and MS16 (encoding a leucine-rich repeat family protein). Cryptosporidium spp. was detected by PCR in 12/352 (3.4%) samples from 4 out of 13 farms. None of the samples tested by microscopy was positive. There was no relationship between Cryptosporidium prevalence and age, sex, diarrhoea or management system; however, Cryptosporidium was found only on farms where horses were kept on pasture during the day and in a stable overnight. Sequence analyses of SSU and gp60 genes revealed the presence of C. muris RN66 (n = 9), Cryptosporidium parvum IIaA15G2R1 (n = 1), Cryptosporidium tyzzeri IXbA22R9 (n = 1), and Cryptosporidium horse genotype VIaA15G4 (n = 1). The C. muris subtypes were identified as MS1-M1, MS2-M4, novel MS2-M7 and MS16-M1 by multilocus sequence of three minisatellite loci. The MS3 locus was not amplified from any isolate. This is the first report of C. tyzzeri and C. muris subtypes from horses.
- MeSH
- Cryptosporidium klasifikace genetika izolace a purifikace MeSH
- feces parazitologie MeSH
- genetická variace * MeSH
- genotyp MeSH
- koně MeSH
- kryptosporidióza epidemiologie MeSH
- nemoci koní parazitologie MeSH
- prevalence MeSH
- průjem parazitologie veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika epidemiologie MeSH
- Polsko epidemiologie MeSH
