Učinak topljivih prehrambenih vlakana na izvedbu vježbi i percepciju umora kod mladih košarkaša

Research background. In this study, we investigated the effects of soluble dietary fibre on improving neuromuscular and cardiovascular endurance and perception of fatigue in a closely monitored group of basketball players. Prebiotics have been sidelined in sports nutrition and their effect on performance remains poorly investigated and understood. Experimental approach. Eighteen healthy male basketball players were divided into two groups; one received 17 g/day of soluble dietary fibre (Nutriose®) for four weeks and the other group received placebo. Their morphological characteristics, neuromuscular and cardiovascular endurance, and rating of perceived exertion according to the rating of perceived exertion (RPE) scale were assessed. Measurements were taken before supplementation and after four weeks of supplementation. Faecal samples were collected from all participants immediately before and after the supplementation period, their total DNA extracted and sent for amplicon sequencing. Results and conclusions. In this study, fibre had no statistically significant effect on the vertical-type explosive power, no statistically significant effect on sprint-type explosive power, nor on aerobic and anaerobic endurance in the experimental group. Soluble fibre had a statistically significant effect on reducing the rating of perceived exertion of basketball players during the competitive part of the season (RPE 7.27±0.04 versus 8.82±0.81). This was confirmed by two-way ANOVA with replication, which showed that within-group interaction (p=0.0193), before and after dietary intake (p=0.0049), and between-group interaction before and after dietary intake (p=0.0313) had a significant effect on the result. The overall conclusion of the study is that soluble dietary fibre supplementation does not improve neuromuscular and cardiovascular endurance over a 4-week period. However, fibre supplementation could have a significant effect on reducing the rating of perceived exertion, as shown by the statistics. Both amplicon sequencing and subsequent bioinformatics results suggest that this could be the result of the beneficial effect on the intestinal microbiota and its metabolites. Novelty and scientific contribution. This work highlights the importance of prebiotics in sports nutrition. Dietary fibre has been a neglected component of sports nutrition. This study demonstrated a statistically significant positive effect on the perception of fatigue, highlighting the need for further studies in this direction.Pozadina istraživanja. U ovom smo radu ispitali učinak topljivih prehrambenih vlakana na poboljšanje neuromuskularne i kardiovaskularne izdržljivosti te smanjenje percepcije umora u pomno praćenoj skupini košarkaša. Prebiotici su zanemarivani u sportskoj prehrani, a njihov učinak na izvedbu vježbi i dalje je slabo istražen i shvaćen. Eksperimentalni pristup. Osamnaest zdravih muških košarkaša podijeljeno je u dvije skupine; jedna je primala 17 g topljivih dijetalnih vlakana (Nutriose®) dnevno tijekom četiri tjedna, a druga je primala placebo. Procijenjene su njihove morfološke karakteristike, neuromuskularna i kardiovaskularna izdržljivost te umor nakon vježbanja prema ljestvici ocjene percipiranog umora. Mjerenja su obavljena na početku i nakon četiri tjedna provedene suplementacije. Uzorci stolice prikupljeni su od svih sudionika neposredno prije i nakon perioda suplementacije, te je iz njih izdvojena ukupna DNK i poslana na sekvenciranje amplikona. Rezultati i zaključci. U ovom istraživanju vlakna nisu imala statistički značajan učinak na eksplozivnu snagu vertikalnog tipa, eksplozivnu snagu sprinta, niti na aerobnu i anaerobnu izdržljivost u eksperimentalnoj skupini. Topljiva vlakna su statistički značajno utjecala na smanjenje ocjene percipiranog umora košarkaša tijekom natjecateljskog dijela sezone (7,27±0,04 naspram 8,82±0,81). To je potvrđeno dvosmjernim ANOVA testom s replikacijom, koji je pokazao da su interakcija unutar skupine (p=0,0193), prije i nakon uzimanja suplementa (p=0,0049) i interakcija između skupina prije i nakon uzimanja suplementa (p=0,0313) značajno utjecale na rezultat. Opći zaključak istraživanja je da dodatak topljivih prehrambenih vlakana ne poboljšava neuromuskularnu i kardiovaskularnu izdržljivost tijekom 4 tjedna. Međutim, suplementacija vlaknima mogla bi imati značajan učinak na smanjenje ocjene percipiranog umora, što je potvrđeno statističkom analizom. Rezultati dobiveni sekvenciranjem amplikona i naknadnom bioinformatičkom obradom sugeriraju da bi to mogao biti ishod blagotvornog učinka suplementa na crijevnu mikrobiotu i njezine metabolite. Novina i znanstveni doprinos. Ovaj rad naglašava važnost uporabe prebiotika u sportskoj prehrani. Prehrambena vlakna su dugo bila zanemarivana komponenta sportske prehrane. Istraživanje je pokazalo statistički značajan pozitivan učinak na percepciju umora, naglašavajući potrebu za daljnjim istraživanjima u tom smjeru

Od sekvencije DNA do kemijske strukture – pretraživanje mikrobnih genomskih i metagenomskih skupova podataka radi pronalaženja novih prirodnih spojeva

Rapid mining of large genomic and metagenomic data sets for modular polyketide synthases, non-ribosomal peptide synthetases and hybrid polyketide synthase/non-ribosomal peptide synthetase biosynthetic gene clusters has been achieved using the generic computer program packages ClustScan and CompGen. These program packages perform the annotation with the hierarchical structuring into polypeptides, modules and domains, as well as storage and graphical presentations of the data. This aims to achieve the most accurate predictions of the activities and specificities of catalytically active domains that can be made with present knowledge, leading to a prediction of the most likely chemical structures produced by these enzymes. The program packages also allow generation of novel clusters by homologous recombination of the annotated genes in silico. ClustScan and CompGen were used to construct a custom database of known compounds (CSDB) and of predicted entirely novel recombinant products (r-CSDB) that can be used for in silico screening with computer aided drug design technology. The use of these programs has been exemplified by analysing genomic sequences from terrestrial prokaryotes and eukaryotic microorganisms, a marine metagenomic data set and a newly discovered example of a \u27shared metabolic pathway\u27 in marine-microbial endosymbiosis.Brzo pretraživanje genomskih i metagenomskih skupova podataka, modularnih biosintetskih genskih nakupina poliketid sintaza i sintetaza neribosomalno sintetiziranih peptida, postignuto je primjenom generičkih računalnih programskih paketa ClustScan i CompGen. Ti programski paketi provode anotaciju hijerarhijskim strukturiranjem podataka na polipeptide, module i domene, te pohranu i grafičku prezentaciju tih podataka. Na temelju dosadašnjih spoznaja, nastoji se postići najtočnije moguće predviđanje aktivnosti i specifičnosti katalitički aktivnih domena, što vodi prema predviđanju najvjerojatnijih kemijskih struktura koje ti enzimi mogu sintetizirati. Programski paketi ClustScan i CompGen omogućuju generiranje novih genskih nakupina homolognom rekombinacijom anotiranih gena u uvjetima in silico, a upotrijebljeni su i za konstrukciju vlastitih baza podataka poznatih poliketidnih i peptidnih supstancija (CSDB) te potpuno novih poliketidnih i peptidnih supstancija produkata rekombinacije (r-CSDB). Ti će se produkti rekombinacije moći upotrijebiti za izbor supstancija s potencijalnom biološkom aktivnošću pomoću računalom vođenog dizajna lijekova u uvjetima in silico. Primjenjivost programskih paketa ClustScan i CompGen dokazana je u analizi genomskih sekvencija prokariotskih i eukariotskih mikroorganizama što žive u tlu, analizi metagenomske skupine podataka u uzorku iz morske vode, a i na nedavno opisanom primjeru \u27zajedničkog metaboličkoga puta\u27 u mikrobnog endosimbionta morske životinje

Od sekvencije DNA do kemijske strukture – pretraživanje mikrobnih genomskih i metagenomskih skupova podataka radi pronalaženja novih prirodnih spojeva

Rapid mining of large genomic and metagenomic data sets for modular polyketide synthases, non-ribosomal peptide synthetases and hybrid polyketide synthase/non-ribosomal peptide synthetase biosynthetic gene clusters has been achieved using the generic computer program packages ClustScan and CompGen. These program packages perform the annotation with the hierarchical structuring into polypeptides, modules and domains, as well as storage and graphical presentations of the data. This aims to achieve the most accurate predictions of the activities and specificities of catalytically active domains that can be made with present knowledge, leading to a prediction of the most likely chemical structures produced by these enzymes. The program packages also allow generation of novel clusters by homologous recombination of the annotated genes in silico. ClustScan and CompGen were used to construct a custom database of known compounds (CSDB) and of predicted entirely novel recombinant products (r-CSDB) that can be used for in silico screening with computer aided drug design technology. The use of these programs has been exemplified by analysing genomic sequences from terrestrial prokaryotes and eukaryotic microorganisms, a marine metagenomic data set and a newly discovered example of a \u27shared metabolic pathway\u27 in marine-microbial endosymbiosis.Brzo pretraživanje genomskih i metagenomskih skupova podataka, modularnih biosintetskih genskih nakupina poliketid sintaza i sintetaza neribosomalno sintetiziranih peptida, postignuto je primjenom generičkih računalnih programskih paketa ClustScan i CompGen. Ti programski paketi provode anotaciju hijerarhijskim strukturiranjem podataka na polipeptide, module i domene, te pohranu i grafičku prezentaciju tih podataka. Na temelju dosadašnjih spoznaja, nastoji se postići najtočnije moguće predviđanje aktivnosti i specifičnosti katalitički aktivnih domena, što vodi prema predviđanju najvjerojatnijih kemijskih struktura koje ti enzimi mogu sintetizirati. Programski paketi ClustScan i CompGen omogućuju generiranje novih genskih nakupina homolognom rekombinacijom anotiranih gena u uvjetima in silico, a upotrijebljeni su i za konstrukciju vlastitih baza podataka poznatih poliketidnih i peptidnih supstancija (CSDB) te potpuno novih poliketidnih i peptidnih supstancija produkata rekombinacije (r-CSDB). Ti će se produkti rekombinacije moći upotrijebiti za izbor supstancija s potencijalnom biološkom aktivnošću pomoću računalom vođenog dizajna lijekova u uvjetima in silico. Primjenjivost programskih paketa ClustScan i CompGen dokazana je u analizi genomskih sekvencija prokariotskih i eukariotskih mikroorganizama što žive u tlu, analizi metagenomske skupine podataka u uzorku iz morske vode, a i na nedavno opisanom primjeru \u27zajedničkog metaboličkoga puta\u27 u mikrobnog endosimbionta morske životinje

Clustering of protein domains for functional and evolutionary studies

Background: The number of protein family members defined by DNA sequencing is usually much larger than those characterised experimentally. This paper describes a method to divide protein families into subtypes purely on sequence criteria. Comparison with experimental data allows an independent test of the quality of the clustering. Results: An evolutionary split statistic is calculated for each column in a protein multiple sequence alignment; the statistic has a larger value when a column is better described by an evolutionary model that assumes clustering around two or more amino acids rather than a single amino acid. The user selects columns (typically the top ranked columns) to construct a motif. The motif is used to divide the family into subtypes using a stochastic optimization procedure related to the deterministic annealing EM algorithm (DAEM), which yields a specificity score showing how well each family member is assigned to a subtype. The clustering obtained is not strongly dependent on the number of amino acids chosen for the motif. The robustness of this method was demonstrated using six well characterized protein families: nucleotidyl cyclase, protein kinase, dehydrogenase, two polyketide synthase domains and small heat shock proteins. Phylogenetic trees did not allow accurate clustering for three of the six families. Conclusion: The method clustered the families into functional subtypes with an accuracy of 90 to 100%. False assignments usually had a low specificity score

Global genome analysis of the shikimic acid pathway reveals greater gene loss in host-associated than in free-living bacteria

<p>Abstract</p> <p>Background</p> <p>A central tenet in biochemistry for over 50 years has held that microorganisms, plants and, more recently, certain apicomplexan parasites synthesize essential aromatic compounds via elaboration of a complete shikimic acid pathway, whereas metazoans lacking this pathway require a dietary source of these compounds. The large number of sequenced bacterial and archaean genomes now available for comparative genomic analyses allows the fundamentals of this contention to be tested in prokaryotes. Using Hidden Markov Model profiles (HMM profiles) to identify all known enzymes of the pathway, we report the presence of genes encoding shikimate pathway enzymes in the hypothetical proteomes constructed from the genomes of 488 sequenced prokaryotes.</p> <p>Results</p> <p>Amongst free-living prokaryotes most Bacteria possess, as expected, genes encoding a complete shikimic acid pathway, whereas of the culturable Archaea, only one was found to have a complete complement of recognisable enzymes in its predicted proteome. It may be that in the Archaea, the primary amino-acid sequences of enzymes of the pathway are highly divergent and so are not detected by HMM profiles. Alternatively, structurally unrelated (non-orthologous) proteins might be performing the same biochemical functions as those encoding recognized genes of the shikimate pathway. Most surprisingly, 30% of host-associated (mutualistic, commensal and pathogenic) bacteria likewise do not possess a complete shikimic acid pathway. Many of these microbes show some degree of genome reduction, suggesting that these host-associated bacteria might sequester essential aromatic compounds from a parasitised host, as a 'shared metabolic adaptation' in mutualistic symbiosis, or obtain them from other consorts having the complete biosynthetic pathway. The HMM results gave 84% agreement when compared against data in the highly curated BioCyc reference database of genomes and metabolic pathways.</p> <p>Conclusions</p> <p>These results challenge the conventional belief that the shikimic acid pathway is universal and essential in prokaryotes. The possibilities that non-orthologous enzymes catalyse reactions in this pathway (especially in the Archaea), or that there exist specific uptake mechanisms for the acquisition of shikimate intermediates or essential pathway products, warrant further examination to better understand the precise metabolic attributes of host-beneficial and pathogenic bacteria.</p

ClustScan: an integrated program package for the semi-automatic annotation of modular biosynthetic gene clusters and in silico prediction of novel chemical structures

The program package ‘ClustScan’ (Cluster Scanner) is designed for rapid, semi-automatic, annotation of DNA sequences encoding modular biosynthetic enzymes including polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS) and hybrid (PKS/NRPS) enzymes. The program displays the predicted chemical structures of products as well as allowing export of the structures in a standard format for analyses with other programs. Recent advances in understanding of enzyme function are incorporated to make knowledge-based predictions about the stereochemistry of products. The program structure allows easy incorporation of additional knowledge about domain specificities and function. The results of analyses are presented to the user in a graphical interface, which also allows easy editing of the predictions to incorporate user experience. The versatility of this program package has been demonstrated by annotating biochemical pathways in microbial, invertebrate animal and metagenomic datasets. The speed and convenience of the package allows the annotation of all PKS and NRPS clusters in a complete Actinobacteria genome in 2–3 man hours. The open architecture of ClustScan allows easy integration with other programs, facilitating further analyses of results, which is useful for a broad range of researchers in the chemical and biological sciences

Bioprospecting for Genes Encoding Hydrocarbon-Degrading Enzymes from Metagenomic Samples Isolated from Northern Adriatic Sea Sediments

Three metagenomic libraries were constructed using surface sediment samples from the northern Adriatic Sea. Two of the samples were taken from a highly polluted and an unpolluted site respectively. The third sample from a polluted site had been enriched using crude oil. The results of the metagenome analyses were incorporated in the REDPET relational database (http://redpet.bioinfo.pbf.hr/REDPET), which was generated using the previously developed MEGGASENSE platform. The database includes taxonomic data to allow the assessment of the biodiversity of metagenomic libraries and a general functional analysis of genes using hidden Markov model (HMM) profiles based on the KEGG database. A set of 22 specialised HMM-profiles was developed to detect putative genes for hydrocarbon-degrading enzymes. Use of these profiles showed that the metagenomic library generated after selection on crude oil had enriched genes for aerobic n-alkane degradation. The use of this system for bioprospecting was exemplified using potential alkB and almA genes from this library

Novi pristup konstrukciji industrijskih mikroorganizama pomoću sintetičke biologije

The recent achievement of synthesising a functioning bacterial chromosome marks a coming of age for engineering living organisms. In the future this should allow the construction of novel organisms to help solve the problems facing the human race, including health care, food, energy and environmental protection. In this minireview, the current state of the field is described and the role of synthetic biology in biotechnology in the short and medium term is discussed. It is particularly aimed at the needs of food technologists, nutritionists and other biotechnologists, who might not be aware of the potential significance of synthetic biology to the research and development in their fields. The potential of synthetic biology to produce interesting new polyketide compounds is discussed in detail.Razvojem područja sinteze funkcionalnog bakterijskog kromosoma obilježen je početak novog doba genetičkog inženjerstva. Konstrukcijom novih organizama mogli bi se riješiti neki problemi vezani uz zdravstvo, proizvodnju hrane i energenata te zaštitu okoliša. U ovom su kratkom revijalnom prikazu opisana sadašnja dostignuća na području sintetičke biologije, a raspravlja se i o njezinoj ulozi u razvoju biotehnologije. Prikaz je posebno namijenjen prehrambenim tehnolozima, nutricionistima i ostalim biotehnolozima koji možda nisu svjesni značaja što bi sintetička biologija mogla imati za njihova istraživanja. Detaljno se raspravlja o mogućem utjecaju sintetičke biologije u formiranju potpuno novih poliketida, koji se mogu upotrijebiti za proizvodnju lijekova

MEGGASENSE – pretraživač (meta)genomski anotiranih sekvencija pomoću govornog jezika – platforma za izradu bioloških skladišta podataka

The MEGGASENSE platform constructs relational databases of DNA or protein sequences. The default functional analysis uses 14 106 hidden Markov model (HMM) profiles based on sequences in the KEGG database. The Solr search engine allows sophisticated queries and a BLAST search function is also incorporated. These standard capabilities were used to generate the SCATT database from the predicted proteome of Streptomyces cattleya. The implementation of a specialised metagenome database (AMYLOMICS) for bioprospecting of carbohydrate-modifying enzymes is described. In addition to standard assembly of reads, a novel ‘functional’ assembly was developed, in which screening of reads with the HMM profiles occurs before the assembly. The AMYLOMICS database incorporates additional HMM profiles for carbohydrate-modifying enzymes and it is illustrated how the combination of HMM and BLAST analyses helps identify interesting genes. A variety of different proteome and metagenome databases have been generated by MEGGASENSE.Platforma MEGGASENSE služi za izradu relacijskih baza podataka koje sadržavaju nukleotidne ili proteinske sekvencije. Osnovna funkcionalna analiza zasniva se na primjeni 14 106 profila skrivenih Markovljevih modela (HMM), temeljenih na sekvencijama dostupnim u bazi podataka KEGG. Pomoću tražilice Solr mogu se zadati napredni upiti u sprezi s implementiranom pretragom BLAST. Osnovne funkcionalnosti platforme omogućile su izradu baze podataka SCATT, temeljene na predviđenom proteomu bakterije Streptomyces cattleya. U radu je opisana implementacija specijalizirane metagenomske baze podataka (AMYLOMICS) za „bioprospecting“ enzima koji modificiraju ugljikohidrate. Uz standardno slaganje očitanih kratkih sljedova DNA, razvijen je funkcionalni postupak pretraživanja HMM profila u očitanim slijedovima DNA prije slaganja. Baza podataka AMYLOMICS sadržava i dodatne HMM profile enzima za modifikaciju ugljikohidrata. U radu je prikazano kako se kombinacijom analiza HMM i BLAST mogu identificirati ciljani geni. Platforma MEGGASENSE upotrijebljena je za izradu raznih proteomskih i metagenomskih baza podataka

Reference-Grade Genome and Large Linear Plasmid of Streptomyces rimosus : Pushing the Limits of Nanopore Sequencing

Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained. IMPORTANCE The genomes of Streptomyces species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids [GLPs]), rearrangements, and high GC content. To improve the quality of the S. rimosus ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters