Quality control in the reaction of aminoacylation

Aminoacil-tRNA-sintetaze (aaRS) su enzimi, prevoditelji genetičkog koda bez kojih proces translacije, pa ni sam život kakav danas poznajemo, ne bi postojao. Omogućavaju pravilno sparivanje aminokiselina i pripadnih molekula tRNA te doprinose visokoj točnosti procesa translacije. Provode raznovrsne mehanizme provjere kontrole kvalitete reakcije aminoaciliranja uključujući i hidrolitičke mehanizme popravka pogrešno aktiviranih aminokiselina i aminoaciliranih tRNA. Važnost popravka pogreške mehanizmom poslije prijenosa aminokiseline na molekulu tRNA (prema engl. posttransfer editing) kojeg provode ovi moćni enzimi pomoću svojih dodatnih domena za popravak, uočava se na primjeru norvalina i meta-tirozina. Popravak pogreške važan je za sprječavanje pogrešne aktivacije i prijenosa nepripadnih proteinogenih aminokiselina na tRNA, ali u zadnje vrijeme se uočava važnost i kod pogrešne aktivacije i prijenosa neproteinogenih aminokiselina na tRNA. Norvalin i meta-tirozin su neproteinogene aminokiseline, analozi leucina i fenilalanina koji predstavljaju veliku prijetnju točnosti procesa translacije i staničnom preživljavanju u uvjetima stresa pogotovo u kompetitivnom okruženju. Mehanizmi popravka pogreške poslije prijenosa efikasno eliminiraju navedene aminokiseline iz genetičkog koda. Proučavanjem mehanizama popravka pogrešaka koje provode aaRS, a koji nisu uvijek i posve evolucijski očuvani, otvara se put razvitku novih antibiotika kojima su bakterijske sintetaze mete, te se omogućava bolje razumijevanje određenih bolesti i razvitak njihovih boljih tretmana.Aminoacyl-tRNA synthetases (aaRS) are enzymes, translators of the genetic code, without which the proces of translation, as well as life as we know it, would not exist. They precisely couple amino acids and cognate tRNA molecules, ensuring in that way a high-fidelity translation. They provide powerful mechanisms of aminoacylation quality control as well as hydrolitic editing (proofreading) of misactivated amino acids and misacylated tRNAs. The importance of posttransfer editing, which takes place within the specific editing domains of aaRS, is obvious in examples of norvaline and meta-tyrosine. Proofreading is of great significance for preventing misactivation and transfer of non-cognate proteinogenic amino acids to tRNAs, but lately its importance in preventing misactivation and transfer of non-proteinogenic amino acids to tRNAs has been noticed. Norvaline and meta-tyrosine are non-proteinogenic amino acids, analogues of leucine and phenylalanine respectively, which pose a significant threat to the accuracy of translation and celullar fitness. Posttransfer editing eliminates effectively both amino acids from the genetic code. Better understanding of the mechanisms of aaRS proofreading opens the door to further development of new antibiotics that target bacterial enzymes, ensures more detailed insight in specific diseases and provides new and possibly more successful medical treatments

Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study

Aerobni anoksigeni fototrofi Jadranskog mora

Aerobic anoxygenic phototrophs are a newly discovered member of the bacterial community in the Adriatic Sea. During the last seven years, when we started to study these organisms, we have collected a considerable number of samples from different environments, from the coast, the estuary, and the open sea. Here we have compiled data from 34 georeferenced study sites from four studies that summarize all that is known about aerobic anoxygenic phototrophs and examine the spatial and vertical distribution and environmental factors affecting this community in the Adriatic Sea. We found horizontal and vertical influences on AAP distribution, mainly salinity, nitrates, chlorophyll a, ammonium, temperature, and soluble reactive phosphorus. Much is known about their ecology in the Adriatic, and with a new survey underway, we will expand our knowledge of their community composition and their role in carbon flux to higher trophic levels.Aerobni anoksigeni fototrofi relativno su novi član bakterijske zajednice u Jadranskom moru. U posljednjih sedam godina, od kada smo započeli proučavati ove organizme, prikupili smo znatan broj uzoraka iz različitih okoliša, obalnog, estuarnog i područja otvorenog mora. U ovom radu objedinili smo podatke s 34 georeferencirane lokacije istraživanja iz četiri studije koje sažimaju sva saznanja o aerobnim anoksigenim fototrofima te istražili prostornu i vertikalnu raspodjelu, kao i okolišne čimbenike koji utječu na ovu zajednicu u Jadranskom moru. Utvrdili smo čimbenike koji utječu na horizontalnu i vertikalnu raspodjelu AAP, uglavnom salinitet, nitrate, klorofil a, amonijeve ione, temperaturu te topljivi reaktivni fosfor. Iako se dosta zna o njihovoj ekologiji u Jadranu, aktualnim istraživanjem proširit ćemo naše znanje o sastavu ove zajednice i njihovoj ulozi u protoku ugljika prema višim trofičkim razinama

Data_Sheet_5_Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.XLSX

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.</p

Data_Sheet_1_Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.DOCX

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.</p

Data_Sheet_2_Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.XLSX

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.</p

Presentation_1_Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.PDF

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.</p

Data_Sheet_3_Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.XLSX

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.</p

Data_Sheet_4_Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.XLSX

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.</p

Association between the SMN2 gene copy number and clinical characteristics of patients with spinal muscular atrophy with homozygous deletion of exon 7 of the SMN1 gene

Background/Aim. Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of alpha motor neurons in the spinal cord and the medulla oblongata, causing progressive muscle weakness and atrophy. The aim of this study was to determine association between the SMN2 gene copy number and disease phenotype in Serbian patients with SMA with homozygous deletion of exon 7 of the SMN1 gene. Methods. The patients were identified using regional Serbian hospital databases. Investigated clinical characteristics of the disease were: patients’ gender, age at disease onset, achieved and current developmental milestones, disease duration, current age, and the presence of the spinal deformities and joint contractures. The number of SMN1 and SMN2 gene copies was determined using real-time polymerase chain reaction (PCR). Results. Among 43 identified patients, 37 (86.0%) showed homozygous deletion of SMN1 exon 7. One (2.7%) of 37 patients had SMA type I with 3 SMN2 copies, 11 (29.7%) patients had SMA type II with 3.1 ± 0.7 copies, 17 (45.9%) patients had SMA type III with 3.7 ± 0.9 copies, while 8 (21.6%) patients had SMA type IV with 4.2 ± 0.9 copies. There was a progressive increase in the SMN2 gene copy number from type II towards type IV (p < 0.05). A higher SMN2 gene copy number was associated with better current motor performance (p < 0.05). Conclusion. In the Serbian patients with SMA, a higher SMN2 gene copy number correlated with less severe disease phenotype. A possible effect of other phenotype modifiers should not be neglected