Optimization of DNA extraction from human urinary samples for mycobiome community profiling.

IntroductionRecent data suggest the urinary tract hosts a microbial community of varying composition, even in the absence of infection. Culture-independent methodologies, such as next-generation sequencing of conserved ribosomal DNA sequences, provide an expansive look at these communities, identifying both common commensals and fastidious organisms. A fundamental challenge has been the isolation of DNA representative of the entire resident microbial community, including fungi.Materials and methodsWe evaluated multiple modifications of commonly-used DNA extraction procedures using standardized male and female urine samples, comparing resulting overall, fungal and bacterial DNA yields by quantitative PCR. After identifying protocol modifications that increased DNA yields (lyticase/lysozyme digestion, bead beating, boil/freeze cycles, proteinase K treatment, and carrier DNA use), all modifications were combined for systematic confirmation of optimal protocol conditions. This optimized protocol was tested against commercially available methodologies to compare overall and microbial DNA yields, community representation and diversity by next-generation sequencing (NGS).ResultsOverall and fungal-specific DNA yields from standardized urine samples demonstrated that microbial abundances differed significantly among the eight methods used. Methodologies that included multiple disruption steps, including enzymatic, mechanical, and thermal disruption and proteinase digestion, particularly in combination with small volume processing and pooling steps, provided more comprehensive representation of the range of bacterial and fungal species. Concentration of larger volume urine specimens at low speed centrifugation proved highly effective, increasing resulting DNA levels and providing greater microbial representation and diversity.ConclusionsAlterations in the methodology of urine storage, preparation, and DNA processing improve microbial community profiling using culture-independent sequencing methods. Our optimized protocol for DNA extraction from urine samples provided improved fungal community representation. Use of this technique resulted in equivalent representation of the bacterial populations as well, making this a useful technique for the concurrent evaluation of bacterial and fungal populations by NGS

Solubility Enhancement of Fe in ZnO Nanoparticles Prepared by Co-Precipitation Method

© 2021, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.Crystalline ZnO offers an excellent host matrix to create a dilute magnetic semiconductor (DMS) owing to its facile Zn-atom substitution with the transition metal dopant atom. The exchange interactions between the spin of the dopant atoms and the carriers in the ZnO matrix results in the room-temperature ferromagnetic order in the entire lattice. In this work, we report on the enhanced solubility (doping) of Fe atoms in ZnO matrix. Zn1-x FexO DMS nanoparticles were synthesized with different doping concentrations (x = 0.01, 0.05, 0.20, 0.22, and 0.25) via a modified version of co-precipitation method, in which the precursors’ solution was heated at 60 ℃ during the stirring process. Only the wurtzite phase was obtained for all Zn1-x FexO samples in X-ray diffraction, and no secondary phase was observed, which supports the idea of an enhanced solubility limit of Fe doping up to 25%. A systematic broadening of the Raman characteristic peak at 525 cm−1 associated with Fe substitution across the entire range of doping accompanied with the suppression of ZnO peak at 371cm−1 and 435 cm−1, supporting the enhanced doping effect further. The bandgap exhibited a systematic trend — it first increased from 3.13 eV for undoped to 3.23 for x = 0.1 and dropped to the value of 2.94 for the highest concentration (x = 0.25) with few in band transitions for high doping. VSM results showed magnetic behavior for all the doped samples at room temperature

Optimization of DNA extraction from human urinary samples for mycobiome community profiling.

IntroductionRecent data suggest the urinary tract hosts a microbial community of varying composition, even in the absence of infection. Culture-independent methodologies, such as next-generation sequencing of conserved ribosomal DNA sequences, provide an expansive look at these communities, identifying both common commensals and fastidious organisms. A fundamental challenge has been the isolation of DNA representative of the entire resident microbial community, including fungi.Materials and methodsWe evaluated multiple modifications of commonly-used DNA extraction procedures using standardized male and female urine samples, comparing resulting overall, fungal and bacterial DNA yields by quantitative PCR. After identifying protocol modifications that increased DNA yields (lyticase/lysozyme digestion, bead beating, boil/freeze cycles, proteinase K treatment, and carrier DNA use), all modifications were combined for systematic confirmation of optimal protocol conditions. This optimized protocol was tested against commercially available methodologies to compare overall and microbial DNA yields, community representation and diversity by next-generation sequencing (NGS).ResultsOverall and fungal-specific DNA yields from standardized urine samples demonstrated that microbial abundances differed significantly among the eight methods used. Methodologies that included multiple disruption steps, including enzymatic, mechanical, and thermal disruption and proteinase digestion, particularly in combination with small volume processing and pooling steps, provided more comprehensive representation of the range of bacterial and fungal species. Concentration of larger volume urine specimens at low speed centrifugation proved highly effective, increasing resulting DNA levels and providing greater microbial representation and diversity.ConclusionsAlterations in the methodology of urine storage, preparation, and DNA processing improve microbial community profiling using culture-independent sequencing methods. Our optimized protocol for DNA extraction from urine samples provided improved fungal community representation. Use of this technique resulted in equivalent representation of the bacterial populations as well, making this a useful technique for the concurrent evaluation of bacterial and fungal populations by NGS