Chromosome Number, Ploidy Level, and Nuclear DNA Content in 23 Species of Echeveria (Crassulaceae)

Echeveria is a polyploid genus with a wide diversity of species and morphologies. The number of species registered for Echeveria is approximately 170; many of them are native to Mexico. This genus is of special interest in cytogenetic research because it has a variety of chromosome numbers and ploidy levels. Additionally, there are no studies concerning nuclear DNA content and the extent of endopolyploidy. This work aims to investigate the cytogenetic characteristics of 23 species of Echeveria collected in 9 states of Mexico, analyzing 2n chromosome numbers, ploidy level, nuclear DNA content, and endopolyploidy levels. Chromosome numbers were obtained from root tips. DNA content was obtained from the leaf parenchyma, which was processed according to the two-step protocol with Otto solutions and propidium iodide as fluorochrome, and then analyzed by flow cytometry. From the 23 species of Echeveria analyzed, 16 species lacked previous reports of 2n chromosome numbers. The 2n chromosome numbers found and analyzed in this research for Echeveria species ranged from 24 to 270. The range of 2C nuclear DNA amounts ranged from 1.26 pg in E. catorce to 7.70 pg in E. roseiflora, while the 1C values were 616 Mbp and 753 Mbp, respectively, for the same species. However, differences in the level of endopolyploidy nuclei were found, corresponding to 4 endocycles (8C, 16C, 32C and 64C) in E. olivacea, E. catorce, E. juarezensis and E. perezcalixii. In contrast, E. longiflora presented 3 endocycles (8C, 16C and 32C) and E. roseiflora presented 2 endocycles (8C and 16C). It has been suggested that polyploidization and diploidization processes, together with the presence of endopolyploidy, allowed Echeveria species to adapt and colonize new adverse environments.

ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples

Background One limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols. Results Using a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols. The improved protocol includes an optimized amplification strategy by using a digital droplet PCR. We demonstrate how PCR products are generated even when using very low concentrated DNA, unable to be detected by using a Qubit. Importantly, the use of different 16S rRNA gene primers had a greater effect on the resulting taxonomical profiles compared to using high or very low initial DNA amounts. Conclusion Our improved protocol takes advantage of ddPCR and allows faithful amplification of very low amounts of template. With this, samples of low bacterial biomass become comparable to those with high amounts of bacteria, since the first and most biasing steps are the same. Besides, it is imperative to state DNA concentrations and volumes used and to include negative controls indicating possible shifts in taxonomical profiles. Despite this, results produced by using different primer pairs cannot be easily compared.

Perfusion-Decellularized Larynx as a Natural 3D Scaffold in a Rabbit Model

<b><i>Introduction:</i></b> Decellularized larynges could be used as scaffolds to regenerate the larynx. The purpose of this study was to establish a perfusion decellularization protocol to produce a 3-dimensional whole laryngeal extracellular matrix (ECM) scaffold in a rabbit model. <b><i>Methods:</i></b> The larynges of 20 rabbits assigned to the study group were harvested and decellularized using a perfusion decellularization protocol, while the larynges of 10 rabbits in the control group were harvested and untreated. Macroscopic and microscopic morphological analyses, a molecular analysis, a cellular content analysis, and scanning electron microscopy were performed. <b><i>Results:</i></b> A histological analysis showed the absence of cellular components, the presence of the ECM, and an intact cartilage structure filled with chondrocytes. The mean total DNA amounts of the native larynx, decellularized larynx, and decellularized cartilage-free larynx were 1,826.40, 434.70, and 41.40 μg/µL, respectively; those for the decellularized larynx and decellularized cartilage-free larynx were significantly lower (<i>p</i> &#x3c; 0.001 and <i>p</i> &#x3c; 0.001, respectively). The total amount of DNA in the decellularized sample was significantly lower compared to that in the native sample, at 57.2% in cartilage (<i>p</i> &#x3c; 0.001), 2.4% in the thyroid gland (<i>p</i> &#x3c; 0.001), 2.7% in muscle (<i>p</i> &#x3c; 0.001), 1.6% in vessels (<i>p</i> &#x3c; 0.001), and 4.8% in the vocal cords (<i>p</i> &#x3c; 0.001). <b><i>Conclusion:</i></b> Our perfusion decellularization protocol is feasible and reproducible to produce a 3-dimensional whole laryngeal ECM scaffold in a rabbit.

Assessment of Bacterial Community Composition and Dynamics in Alfalfa Silages With and Without Lactobacillus plantarum Inoculation Using Absolute Quantification 16S rRNA Sequencing

Relative quantification 16S-seq (RQS) has drawn deeper insights into bacterial community compositions in silage. However, it provides no information on dynamics of the total amount of bacterial DNA through the ensiling process and across different treatments. In this study, bacterial compositions in alfalfa silage with and without Lactobacillus plantarum inoculation after 10 and 60days of ensiling were investigated using absolute quantification 16S-seq (AQS), and bacterial composition and its interaction with fermentation properties of silage indicated by AQS and RQS were compared. Variation in total bacterial DNA amounts across different treatments and ensiling periods was illustrated by AQS. AQS indicated higher bacterial richness indices and closer correlations of these indices with fermentation properties than RQS via spearman’s correlation analyses, as well as more taxa with significance on bacterial abundance via lefse analyses. In conclusion, AQS effectively illustrated the dynamics of bacterial communities during the ensiling process.

Composition and Predominance of Fusarium Species Causing Fusarium Head Blight in Winter Wheat Grain Depending on Cultivar Susceptibility and Meteorological Factors

Fusarium head blight (FHB) is one of the most important diseases of wheat, causing yield losses and mycotoxin contamination of harvested grain. A complex of different toxigenic Fusarium species is responsible for FHB and the composition and predominance of species within the FHB complex are determined by meteorological and agronomic factors. In this study, grain of three different susceptible winter wheat cultivars from seven locations in northern Germany were analysed within a five-year survey from 2013 to 2017 by quantifying DNA amounts of different species within the Fusarium community as well as deoxynivalenol (DON) and zearalenone (ZEA) concentrations. Several Fusarium species co-occur in wheat grain samples in all years and cultivars. F. graminearum was the most prevalent species, followed by F. culmorum, F. avenaceum and F. poae, while F. tricinctum and F. langsethiae played only a subordinate role in the FHB complex in terms of DNA amounts. In all cultivars, a comparable year-specific quantitative occurrence of the six detected species and mycotoxin concentrations were found, but with decreased DNA amounts and mycotoxin concentrations in the more tolerant cultivars, especially in years with higher disease pressure. In all years, similar percentages of DNA amounts of the six species to the total Fusarium DNA amount of all detected species were found between the three cultivars for each species, with F. graminearum being the most dominant species. Differences in DNA amounts and DON and ZEA concentrations between growing seasons depended mainly on moisture factors during flowering of wheat, while high precipitation and relative humidity were the crucial meteorological factors for infection of wheat grain by Fusarium. Highly positive correlations were found between the meteorological variables precipitation and relative humidity and DNA amounts of F. graminearum, DON and ZEA concentrations during flowering, whereas the corresponding correlations were much weaker several days before (heading) and after flowering (early and late milk stage).

Quantum patterns of genome size variation in angiosperms

Aims: The discontinuous pattern of genome size variation in angiosperms is an unsolved problem related to genome evolution. We introduce a genome evolution operator and solve the related eigen-value equation to deduce the discontinuous pattern. Background: Genome is a well-defined system for studying evolution of species. One of the basic problems is the genome size evolution. The DNA amounts for angiosperm species are highly variable differing over 1000-fold. One big surprise is the discovery of the discontinuous distribution of nuclear DNA amounts in many angiosperm genera. Objective: The discontinuous distribution of nuclear DNA amounts have certain regularity much like a group of quantum states in atomic physics. The quantum pattern has not been explained by all the evolutionary theories so far and we shall interpret it through the quantum simulation of genome evolution. Methods: We have introduced a genome evolution operator H to deduce the distribution of DNA amount. The nuclear DNA amount in angiosperms is studied from the eigen-value equation of the genome evolution operator H. The operator H is introduced by physical simulation and it is defined as a function of the genome size N and the derivative with respective to the size. Results: The discontinuity of DNA size distribution and its synergetic occurrence in related angiosperms species are successfully deduced from the solution of the equation. The results agree well with the existing experimental data of Aloe, Clarkia, Nicotiana, Lathyrus, Allium and other genera. Conclusion: The success of our approach may infer the existence of a set of genomic evolutionary equations satisfying classical – quantum duality. The classical phase of evolution means it obeying classical deterministic law, while the quantum phase means it obeying quantum stochastic law. The discontinuity of DNA size distribution provides fresh evidence on the quantum evolution of angiosperms. People realize that the discontinuous pattern is due to the existence of some unknown evolutionary constrains. However, our study indicates that these constrains on angiosperm genome are essentially of quantum origin.

Statistical modeling of STR capillary electrophoresis signal

Background In order to isolate an individual’s genotype from a sample of biological material, most laboratories use PCR and Capillary Electrophoresis (CE) to construct a genetic profile based on polymorphic loci known as Short Tandem Repeats (STRs). The resulting profile consists of CE signal which contains information about the length and number of STR units amplified. For samples collected from the environment, interpretation of the signal can be challenging given that information regarding the quality and quantity of the DNA is often limited. The signal can be further compounded by the presence of noise and PCR artifacts such as stutter which can mask or mimic biological alleles. Because manual interpretation methods cannot comprehensively account for such nuances, it would be valuable to develop a signal model that can effectively characterize the various components of STR signal independent of a priori knowledge of the quantity or quality of DNA. Results First, we seek to mathematically characterize the quality of the profile by measuring changes in the signal with respect to amplicon size. Next, we examine the noise, allele, and stutter components of the signal and develop distinct models for each. Using cross-validation and model selection, we identify a model that can be effectively utilized for downstream interpretation. Finally, we show an implementation of the model in NOCIt, a software system that calculates the a posteriori probability distribution on the number of contributors. Conclusion The model was selected using a large, diverse set of DNA samples obtained from 144 different laboratory conditions; with DNA amounts ranging from a single copy of DNA to hundreds of copies, and the quality of the profiles ranging from pristine to highly degraded. Implemented in NOCIt, the model enables a probabilisitc approach to estimating the number of contributors to complex, environmental samples.

Inter-laboratory study on standardized MPS libraries: evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA

We present results from an inter-laboratory massively parallel sequencing (MPS) study in the framework of the SeqForSTRs project to evaluate forensically relevant parameters, such as performance, concordance, and sensitivity, using a standardized sequencing library including reference material, mixtures, and ancient DNA samples. The standardized library was prepared using the ForenSeq DNA Signature Prep Kit (primer mix A). The library was shared between eight European laboratories located in Austria, France, Germany, The Netherlands, and Sweden to perform MPS on their particular MiSeq FGx sequencers. Despite variation in performance between sequencing runs, all laboratories obtained quality metrics that fell within the manufacturer’s recommended ranges. Furthermore, differences in locus coverage did not inevitably adversely affect heterozygous balance. Inter-laboratory concordance showed 100% concordant genotypes for the included autosomal and Y-STRs, and still, X-STR concordance exceeded 83%. The exclusive reasons for X-STR discordances were drop-outs at DXS10103. Sensitivity experiments demonstrated that correct allele calling varied between sequencing instruments in particular for lower DNA amounts (≤ 125 pg). The analysis of compromised DNA samples showed the drop-out of one sample (FA10013B01A) while for the remaining three degraded DNA samples MPS was able to successfully type ≥ 87% of all aSTRs, ≥ 78% of all Y-STRs, ≥ 68% of all X-STRs, and ≥ 92% of all iSNPs demonstrating that MPS is a promising tool for human identity testing, which in return, has to undergo rigorous in-house validation before it can be implemented into forensic routine casework.

2-Methacryloyloxyethyl phosphorylcholine (MPC)-polymer suppresses an increase of oral bacteria: a single-blind, crossover clinical trial

Objectives The biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymers, which mimic a biomembrane, reduce protein adsorption and bacterial adhesion and inhibit cell attachment. The aim of this study is to clarify whether MPC-polymer can suppress the bacterial adherence in oral cavity by a crossover design. We also investigated the number of Fusobacterium nucleatum, which is the key bacterium forming dental plaque, in clinical samples. Materials and methods This study was a randomized, placebo-controlled, single-blind, crossover study, with two treatment periods separated by a 2-week washout period. We conducted clinical trial with 20 healthy subjects to evaluate the effect of 5% MPC-polymer mouthwash after 5 h on oral microflora. PBS was used as a control. The bacterial number in the gargling sample before and after intervention was counted by an electronic bacterial counter and a culture method. DNA amounts of total bacteria and F. nucleatum were examined by q-PCR. Results The numbers of total bacteria and oral streptcocci after 5 h of 5% MPC-polymer treatment significantly decreased, compared to the control group. Moreover, the DNA amounts of total bacteria and F. nucleatum significantly decreased by 5% MPC-polymer mouthwash. Conclusions We suggest that MPC-polymer coating in the oral cavity may suppress the oral bacterial adherence. Clinical relevance MPC-polymer can be a potent compound for the control of oral microflora to prevent oral infection.