Dedicated transcriptomics combined with power analysis lead to functional understanding of genes with weak phenotypic changes in knockout lines

Author summary Knockout mice benefit the understanding of gene functions in mammals. However, it has proven difficult for many genes to identify clear phenotypes, related due to lack of sufficient assays. As Lewis Wolpert put it in a famous quote “But did you take them to the opera?”, thus metaphorically alluding to the need to extend phenotyping efforts. This insight led to the establishment of phenotyping pipelines that are nowadays routinely used to characterize knock-out lines. However, transcriptomic approaches based on RNA-Seq have been much less explored for such deep-level studies. We conducted here both, a theoretical power analysis and practical RNA-Seq experiments on two knockout lines with small phenotypic effects to investigate the parameters including sample size, sequencing depth, fold change, and dispersion. Our dedicated RNA-Seq studies discovered thousands of genes with small transcriptional changes and enriched in specific functions in both knockout lines. We find that it is more important to increase the number of samples than to increase the sequencing depth. Our work shows that a deep RNA-Seq study on knockouts is powerful for understanding gene functions in cases of weak phenotypic effects, and provides a guideline for the experimental design of such studies

Diatom metabarcoding and microscopic analyses from sediment samples at Lake Nam Co, Tibet: The effect of sample-size and bioinformatics on the identified communities

Diatoms (Bacillariophyceae) are characterized by silicified cell walls that favor their long-term preservation in sediments, therefore widely used as bioindicators of present and past water conditions. Alongside with traditional morphological analyses, metabarcoding has become a valuable tool to study the community structures of various organisms, including diatoms. Here, we test whether the quantity of sediment sample used for DNA extraction affects the results obtained from high-throughput sequencing (metabarcoding) of the diatom rbcL region by isolating DNA from 10 g and 0.5 g (wet weight) of lake surface sediment samples. Because bioinformatics processing of metabarcoding data may affect the outcome, we also tested the consistency of the results from three different pipelines: 1) ESVs (exact sequence variants) pipeline; 2) clustering sequences at 95% sequence identity to form OTUs (operational taxonomic units; 95% OTUs); and 3) 97% OTUs pipeline. Additionally, the agreement between metabarcoding data and morphological inventories of corresponding samples were compared. Our results demonstrate highly uniform patterns between the diatom rbcL amplicons from 10 g and 0.5 g of sedimentary DNA (sedDNA) extracts (HTS 10 and HTS 0.5, respectively). Furthermore, after the careful curation of the sequencing data, metabarcoding results were highly consistent among the data sets produced by different bioinformatics pipelines. Comparing results from metabarcoding and microscopy, we identified some taxonomic mismatches: morphological analyses identified 59 diatom genera, whereas metabarcoding 49 to 54 genera. These mismatches are related to incompleteness of the sequence databases, but also to inconsistencies in diatom taxonomy in general and potential dissolution effects of diatom valves caused by high alkalinity of the investigated lake waters. Nevertheless, multivariate community analysis revealed consistent results between data sets identified by microscopy and metabarcoding – water depth and conductivity as the most significant variables in driving diatom communities in Lake Nam Co – further confirming that metabarcoding is a viable method for identifying diatom-environment relationships

Characterization of the microbiome of the invasive Asian toad in Madagascar across the expansion range and comparison with a native co-occurring species

Biological invasions are on the rise, with each invader carrying a plethora of associated microbes. These microbes play important, yet poorly understood, ecological roles that can include assisting the hosts in colonization and adaptation processes or as possible pathogens. Understanding how these communities differ in an invasion scenario may help to understand the host's resilience and adaptability. The Asian common toad, Duttaphrynus melanostictus is an invasive amphibian, which has recently established in Madagascar and is expected to pose numerous threats to the native ecosystems. We characterized the skin and gut bacterial communities of D. melanostictus in Toamasina (Eastern Madagascar), and compared them to those of a co-occurring native frog species, Ptychadena mascareniensis, at three sites where the toad arrived in different years. Microbial composition did not vary among sites, showing that D. melanostictus keeps a stable community across its expansion but significant differences were observed between these two amphibians. Moreover, D. melanostictus had richer and more diverse communities and also harboured a high percentage of total unique taxa (skin: 80%; gut: 52%). These differences may reflect the combination of multiple host-associated factors including microhabitat selection, skin features and dietary preferences

Improved threshold of buried heterostructure InAs/GaInAsP quantum dot lasers

This article may be downloaded for personal use only. Any other use requires prior permission of the author and AIP Publishing. This article appeared in Journal of Applied Physics 109, 083104 (2011) and may be found at https://doi.org/10.1063/1.3574406.The parameters for reducing the threshold current density of InAs/InGaAsP/InP quantum-dot (QD) lasers suitable for high temperature operation are studied. The structures were grown using metalorganic vapor phase epitaxy. Increasing the number of QD layers leads to a substantial improvement of the optical confinement and a markedly reduced threshold per dot layer in broad area devices. A reduction of the spacer thickness between the QD layers was not found to significantly affect device characteristics. Depending upon the device length, an optimum number of QD layers was deduced. Based upon optimized QD stacks, buried-heterostructure lasers with a medium device length emitting at 1.5 μm were fabricated. Laterally single-mode devices show promising low threshold currents near 10 mA and good thermal stability with a characteristic temperature of 65 K up to 90 °C.DFG, 43659573, SFB 787: Halbleiter - Nanophotonik: Materialien, Modelle, Bauelement

Dogs as carriers of virulent and resistant genotypes of Clostridioides difficile

Abstract While previous research on zoonotic transmission of community-acquired Clostridioides difficile infection (CA-CDI) focused on food-producing animals, the present study aimed to investigate whether dogs are carriers of resistant and/or virulent C. difficile strains. Rectal swabs were collected from 323 dogs and 38 C. difficile isolates (11.8%) were obtained. Isolates were characterized by antimicrobial susceptibility testing, whole-genome sequencing (WGS) and a DNA hybridization assay. Multilocus sequence typing (MLST), core genome MLST (cgMLST) and screening for virulence and antimicrobial resistance genes were performed based on WGS. Minimum inhibitory concentrations for erythromycin, clindamycin, tetracycline, vancomycin and metronidazole were determined by E-test. Out of 38 C. difficile isolates, 28 (73.7%) carried genes for toxins. The majority of isolates belonged to MLST sequence types (STs) of clade I and one to clade V. Several isolates belonged to STs previously associated with human CA-CDI. However, cgMLST showed low genetic relatedness between the isolates of this study and C. difficile strains isolated from humans in Austria for which genome sequences were publicly available. Four isolates (10.5%) displayed resistance to three of the tested antimicrobial agents. Isolates exhibited resistance to erythromycin, clindamycin, tetracycline and metronidazole. These phenotypic resistances were supported by the presence of the resistance genes erm(B), cfr(C) and tet(M). All isolates were susceptible to vancomycin. Our results indicate that dogs may carry virulent and antimicrobial-resistant C. difficile strains.1 Introduction 2 Methods 2.1 Sampling and ethics 2.2 Isolation and identification of Clostridioides difficile 2.3 Antimicrobial susceptibility testing 2.4 Whole-genome sequencing and comparative genomic analysis 2.5 Statistical analysis 3 Results 3.1 Prevalence of Clostridioides difficile and risk factors for shedding 3.2 Antimicrobial susceptibility testing and detection of antimicrobial resistance determinants 3.3 Genomic characterization of canine Clostridioides difficile 3.4 Genome annotation and comparison 4 Discussio

Evolution and Flare Activity of Delta-Sunspots in Cycle 23

The emergence and magnetic evolution of solar active regions (ARs) of beta-gamma-delta type, which are known to be highly flare-productive, were studied with the SOHO/MDI data in Cycle 23. We selected 31 ARs that can be observed from their birth phase, as unbiased samples for our study. From the analysis of the magnetic topology (twist and writhe), we obtained the following results. i) Emerging beta-gamma-delta ARs can be classified into three topological types as "quasi-beta", "writhed" and "top-to-top". ii) Among them, the "writhed" and "top-to-top" types tend to show high flare activity. iii) As the signs of twist and writhe agree with each other in most cases of the "writhed" type (12 cases out of 13), we propose a magnetic model in which the emerging flux regions in a beta-gamma-delta AR are not separated but united as a single structure below the solar surface. iv) Almost all the "writhed"-type ARs have downward knotted structures in the mid portion of the magnetic flux tube. This, we believe, is the essential property of beta-gamma-delta ARs. v) The flare activity of beta-gamma-delta ARs is highly correlated not only with the sunspot area but also with the magnetic complexity. vi) We suggest that there is a possible scaling-law between the flare index and the maximum umbral area

Linkage map construction involving a reciprocal translocation

This paper is concerned with a novel statistical–genetic approach for the construction of linkage maps in populations obtained from reciprocal translocation heterozygotes of barley (Hordeum vulgare L.). Using standard linkage analysis, translocations usually lead to ‘pseudo-linkage’: the mixing up of markers from the chromosomes involved in the translocation into a single linkage group. Close to the translocation breakpoints recombination is severely suppressed and, as a consequence, ordering markers in those regions is not feasible. The novel strategy presented in this paper is based on (1) disentangling the “pseudo-linkage” using principal coordinate analysis, (2) separating individuals into translocated types and normal types and (3) separating markers into those close to and those more distant from the translocation breakpoints. The methods make use of a consensus map of the species involved. The final product consists of integrated linkage maps of the distal parts of the chromosomes involved in the translocation

Caries prevalence and tooth loss in Hungarian adult population: results of a national survey

<p>Abstract</p> <p>Background</p> <p>Oral health is basicly important for the well-being of people. Thus, it is strongly suggested to organize epidemiological surveys in order to gain representative data on oral condition of the given population. The purpose of the cross-sectional study was to determine the results on tooth loss and caries prevalence of Hungarian adults in different age groups.</p> <p>Methods</p> <p>Altogether 4606 persons (2923 women, 1683 men) participated in the study who were classified into different age groups: 19 [less than or equal to], 20–24, 35–44, 45–64, 65–74, [greater than or equal to]75 year olds. Probands were selected randomly from the population attending the compulsory lung screening examinations. The participants were examined by calibrated dentists, according to the WHO (1997) criteria. In order to produce representative data, the chosen localities for these examinations covered the capital, the largest towns, the villages, and case weights were used for the statistical evaluation.</p> <p>Results</p> <p>The mean values of DMF-T were found between 11.79±5.68 and 21.90±7.61 These values were significantly higher in women compared to men (p < 0.05). In all age groups the values of M were the highest. Except for the women in the groups of 35–44 and 45–64 year olds, these values showed an increasing tendency both in women and men by age (from 5.50±6.49, and 4.70±4.08 to 21.52±9.07 and 18.41±8.89 respectively). The values of D components reached the highest values in 45–64 year olds (4.54±2.12 and 4.22±2.81, by gender, respectively), then in the older age groups there was a high reduction in these values (in 65–74 year olds: 2.72±1.88 and 1.36±2.48; in 75 or more than 75 year olds: 1.05±1.41 and 1.03±1.76 by gender, respectively). The ratio of D and F values was the highest in the age group of 65–74 year olds (2.12), the lowest ratio could be calculated in 20–34 year olds (0.65).</p> <p>Data showed some decrease in caries experience in 35–44 years of age between 2000 and 2004. The prevalence of persons with 21 or more teeth had been increased from 65.6% to 73.1%. This positive tendency has not been occured in prevalence of edentulousness in this age group: the prevalence of edentulous persons changed from 1.4 to 1.9%. In 65–74 year olds the level of edentulousness became lower, from 25.9 to 14.8% and the prevalence of persons with 21 or more teeth is higher (22.6%) than it was in 2000 (13.0%).</p> <p>Conclusion</p> <p>Present data from Hungary show some slight decrease in caries experience between 35–44 years of age, although this positive tendency has not been occured in prevalence of edentulousness in this age group. A positive tendency could be experienced in the group of 65–74 year olds in edentulousness and in number of teeth, but further efforts are needed to reach a better situation.</p