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Joran Martijn Anders E. Lind Max E. Schön Ian Spiertz Lina Juzokaite Ignas Bunikis Olga V. Pettersson Thijs J. G. Ettema 《Environmental microbiology》2019,21(7):2485-2498
Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput. 相似文献
3.
Lina Long Douglas R. McCabe M. Eileen Dolan 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,731(2):128
A highly sensitive and selective method for determining 8-oxoguanine in plasma and urine was developed by high-performance liquid chromatography with electrochemical detection. The compound was separated by gradient elution on a C18 reversed-phase column with a mobile phase of acetonitrile and 0.1 M sodium acetate, pH 5.2. 8-Hydroxy-2′-deoxyguanosine was used as internal standard. 8-Oxoguanine was detected electrochemically by setting the potential to +300 mV vs. Pd reference. The sensitivity of the assay was 22 ng/ml with a signal-to-noise ratio of 7:1. The within-day relative standard deviations for 8-oxoguanine quality control samples with concentrations of 3340, 1340 and 84 ng/ml were 3.6, 4.3 and 5.7% for plasma, and 4.1, 4.6 and 6.2% for urine, respectively. The day-to-day relative standard deviations for the same samples were 3.8, 6.8 and 7.1% for plasma, and 3.9, 7.0 and 7.9% for urine, respectively. The method is designed to study the pharmacokinetics and metabolic fate of O6-benzylguanine in a phase I clinical trial. Previously, O6-benzyl-8-oxoguanine was identified as the primary metabolite of O6-benzylguanine in humans. We now demonstrate that 8-oxoguanine is a further metabolite of O6-benzylguanine. 相似文献
4.
Ross A. Robinson Samuel C. Griffiths Lieke L. van de Haar Tomas Malinauskas Eljo Y. van Battum Pavol Zelina Rebekka A. Schwab Dimple Karia Lina Malinauskaite Sara Brignani Marleen H. van den Munkhof Özge Düdükcü Anna A. De Ruiter Dianne M.A. Van den Heuvel Benjamin Bishop Jonathan Elegheert A. Radu Aricescu R. Jeroen Pasterkamp Christian Siebold 《Cell》2021,184(8):2103-2120.e31
5.
Lina Zhang Changyong Zhou Qiaoji Qin Zhenfang Liu Peng Li 《Journal of cellular biochemistry》2019,120(9):14670-14678
Long noncoding RNAs (lncRNAs) play important roles in endothelium development. A lncRNA, LEF1-AS1, is recently emerging as a potent mediator of the proliferation and migration of a number of cells, including smooth muscle cells. However, the effects of LEF1-AS1 in atherosclerosis remains largely unknown. Specimens from patients with coronary artery atherosclerosis were collected. The quantitative real-time polymerase chain reaction was used to analyze levels of LEF1-AS1 and microRNA-544a (miR-544a). Western blot analysis was used to assess PTEN, P-Akt, and T-Akt protein expression. Proliferation, migration, and invasion of cells were analyzed by cell counting kit-8 assay, scratch wound assay, and transwell assay, respectively. The interaction between LEF1-AS1, miR-544a, and PTEN was probed using bioinformatical analysis and dual-luciferase assay. In plasma and tissue of patients with coronary artery atherosclerosis, LEF1-AS1 was upregulated and miR-544a was downregulated. A negative correlation was found between LEF1-AS1 and miR-544a. miR-544a overexpression reversed the inhibition of LEF1-AS1 in smooth muscle cell proliferation and invasion, which were mediated through the PTEN pathway. LEF1-AS1 regulates smooth muscle cell proliferation and migration through the miR-544a/PTEN axis, indicating that LEF1-AS1 may be a potential therapeutic target in atherosclerosis. 相似文献
6.
Isolation of viral coat protein mutants with altered assembly and aggregation properties 总被引:1,自引:0,他引:1 下载免费PDF全文
A method was developed to screen bacteria for synthesis of mutant proteins with altered assembly and solubility properties using bacteriophage MS2 coat protein as a model self-associating protein. Colonies expressing coat protein from a plasmid were covered with an agarose overlay under conditions that caused the lysis of some of the cells in each colony. The proteins thus liberated diffused through the overlay at rates depending on their molecular sizes. After transfer of the proteins to a nitrocellulose membrane, probing with coat protein-specific antiserum revealed spots whose sizes and intensities were related to the aggregation state of coat protein. The method was employed in the isolation of assembly defective mutants and to find soluble variants of an aggregation-prone coat protein mutant. 相似文献
7.
Montserrat Barriga Roi Cal Nuria Cabello Anna Llach Alexander Vallmitjana Raúl Benítez Lina Badimon Juan Cinca Vicenta Llorente-Cortés Leif Hove-Madsen 《PloS one》2013,8(3)
The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 µg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 µg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 µg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 µg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal. 相似文献
8.
Lina L. E. Massone 《Biological cybernetics》1994,71(4):293-305
This paper presents a neural-network-based system that can generate and control movements of the eyes. It was inspired by a number of experimental observations on the saccadic and gaze systems of monkeys and cats. Because of the generality of the approach undertaken, the system can be regarded as a demonstration of how parallel distributed processing principles, namely learning and attractor dynamics, can be integrated with experimental findings, as well as a biologically inspired controller for a dexterous robotic orientation device. The system is composed of three parts: a dynamic motor map, a push-pull circuitry, and a plant. The dynamics of the motor map is generated by a multi-layer network that was trained to compute a bidimensional temporal-spatial transformation. Simulation results indicate (1) that the system is able to reproduce some of the properties observed in the biological system at the neural and movement levels and (2) that the dynamics of the motor map remains stereotyped even when the motor map is subject to abnormal stimulation patterns. The latter result emphasizes the role of the topographic projection that connects the motor map to the push-pull circuitry in determining the features of the resulting movements. 相似文献
9.
Lina J. Bird Maureen L. Coleman Dianne K. Newman 《Applied and environmental microbiology》2013,79(12):3619-3627
Transition metals are known to cause toxic effects through their interaction with oxygen, but toxicity under anoxic conditions is poorly understood. Here we investigated the effects of iron (Fe) and copper (Cu) on the anaerobic growth and gene expression of the purple phototrophic bacterium Rhodopseudomonas palustris TIE-1. We found that Fe(II) and Cu(II) act synergistically to delay anaerobic growth at environmentally relevant metal concentrations. Cu(I) and Cu(II) had similar effects both alone and in the presence of ascorbate, a Cu(II) reductant, indicating that reduction of Cu(II) to Cu(I) by Fe(II) is not sufficient to explain the growth inhibition. Addition of Cu(II) increased the toxicity of Co(II) and Ni(II); in contrast, Ni(II) toxicity was diminished in the presence of Fe(II). The synergistic anaerobic toxicity of Fe(II) and Cu(II) was also observed for Escherichia coli MG1655, Shewanella oneidensis MR-1, and Rhodobacter capsulatus SB1003. Gene expression analyses for R. palustris identified three regulatory genes that respond to Cu(II) and not to Fe(II): homologs of cueR and cusR, two known proteobacterial copper homeostasis regulators, and csoR, a copper regulator recently identified in Mycobacterium tuberculosis. Two P-type ATPase efflux pumps, along with an FoF1 ATP synthase, were also upregulated by Cu(II) but not by Fe(II). An Escherichia coli mutant deficient in copA, cus, and cueO showed a smaller synergistic effect, indicating that iron might interfere with one or more of the copper homeostasis systems. Our results suggest that interactive effects of transition metals on microbial physiology may be widespread under anoxic conditions, although the molecular mechanisms remain to be more fully elucidated. 相似文献