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Background The great diversity in plant genome size and chromosome number is partly due to polyploidization (i.e. genome doubling events). The differences in genome size and chromosome number among diploid plant species can be a window into the intriguing phenomenon of past genome doubling that may be obscured through time by the process of diploidization. The genus Hibiscus L. (Malvaceae) has a wide diversity of chromosome numbers and a complex genomic history. Hibiscus is ideal for exploring past genomic events because although two ancient genome duplication events have been identified, more are likely to be found due to its diversity of chromosome numbers. To reappraise the history of whole-genome duplication events in Hibiscus, we tested three alternative scenarios describing different polyploidization events. Results Using target sequence capture, we designed a new probe set for Hibiscus and generated 87 orthologous genes from four diploid species. We detected paralogues in > 54% putative single-copy genes. 34 of these genes were selected for testing three different genome duplication scenarios using gene counting. All species of Hibiscus sampled shared one genome duplication with H. syriacus, and one whole genome duplication occurred along the branch leading to H. syriacus. Conclusions Here, we corroborated the independent genome doubling previously found in the lineage leading to H. syriacus and a shared genome doubling of this lineage and the remainder of Hibiscus. Additionally, we found a previously undiscovered genome duplication shared by the /Pavonia and /Malvaviscus clades (both nested within Hibiscus) with the occurrences of two copies in what were otherwise single-copy genes. Our results highlight the complexity of genomic diversity in some plant groups, which makes orthology assessment and accurate phylogenomic inference difficult.

When rainwater harvesting is utilized as an alternative water resource in buildings, a combination of municipal water and rainwater is typically required to meet water demands. Altering source water chemistry can disrupt pipe scale and biofilm and negatively impact water quality at the distribution level. Still, it is unknown if similar reactions occur within building plumbing following a transition in source water quality. The goal of this study was to investigate changes in water chemistry and microbiology at a green building following a transition between municipal water and rainwater. We monitored water chemistry (metals, alkalinity, and disinfectant byproducts) and microbiology (total cell counts, plate counts, and opportunistic pathogen gene markers) throughout two source water transitions. Several constituents including alkalinity and disinfectant byproducts served as indicators of municipal water remaining in the system since the rainwater source does not contain these constituents. In the treated rainwater, microbial proliferation and Legionella spp. gene copy numbers were often three logs higher than those in municipal water. Because of differences in source water chemistry, rainwater and municipal water uniquely interacted with building plumbing and generated distinctively different drinking water chemical and microbial quality profiles.

A quantitative real-time polymerase chain reaction (QPCR) was applied to estimate biokinetic coefficients of Clostridium cadaveris and Clostridium sporogenes, which utilize protein as carbon source. Experimental data of changes in peptone concentration and 16S rRNA gene copy numbers of C. cadaveris and C. sporogenes were fitted to model. The fourth-order Runge-Kutta approximation with non-linear least squared analysis was employed to solve the ordinary differential equations to estimate biokinetic coefficients. The maximum specific growth rate (μmax), half saturation concentration (Ks), growth yield (Y), and decay coefficient (Kd) of C. cadaveris and C.sporogenes were 0.73 ± 0.05 and 1.35 ± 0.32 h-1, 6.07 ± 1.52 and 5.67 ± 1.53 g/L, 2.25 ± 0.75 × 1010 and 7.92 ± 3.71 × 109 copies/g, 0.002 ± 0.003 and 0.002 ± 0.001 h-1, respectively. The theoretical specific growth rate of C. sporogenes always exceeded than that of C. cadaveris at peptone concentration higher than 3.62 g/L. When the influent peptone concentration was 5.0 g/L, the concentration of C.cadaveris gradually decreased to the steady value of 2.9 × 1010 copies/mL at 4 hours HRT, which indicates 67.1% of the initial population reduction, but the wash out occurred at 1.9 and 3.2 hours HRTs. The 16S rRNA gene copy numbers of C. sporogenes gradually decreased to steady values ranging from 1.1 × 1010 to 2.9 × 1010 copies/mL. C. sporogenes species was predicted to wash out at an HRT of 1.6 h.

Wastewater-based surveillance is a cost-effective concept for monitoring COVID-19 pandemics at a population level. Here, SARS-CoV-2 RNA was monitored from a total of 693 wastewater (WW) influent samples from 28 wastewater treatment plants (WWTP, N = 21–42 samples per WWTP) in Finland from August 2020 to May 2021, covering WW of ca. 3.3 million inhabitants (∼ 60% of the Finnish population). The relative quantity of SARS-CoV-2 RNA fragments in the 24h-composite samples was determined by using the ultrafiltration method followed by nucleic acid extraction and RT-qPCR assay targeted with N2-assay. SARS-CoV-2 RNA signals at each WWTP were compared over time to the numbers of new and confirmed COVID-19 cases in the sewer network area.Over the 10-month surveillance period, the detection rate of SARS-CoV-2 RNA in WW was 79% (including 6% uncertain results), while only 24% of all samples exhibited gene copy (GC) numbers above the quantification limit. The range of the SARS-CoV-2 detection rate in WW varied from 33% (including 10% uncertain results) in Pietarsaari to 100% in Espoo. Only six out of 693 WW samples were positive with SARS-COV-2 RNA when the reported COVID-19 case number from the preceding 14 days was zero. Overall, the 14-day COVID-19 incidence was 7, 18 and 36 cases within the sewer network area when the probability to detect SARS-CoV-2 RNA in wastewater samples was 50%, 75% and 95%, respectively. The quantification of SARS-CoV-2 GC required significantly more COVID-19 cases: the quantification rate was 50%, 75% and 95% when the 14-day incidence was 110, 152 and 223 COVID-19 cases, respectively, per 100 000 persons. Multiple linear regression confirmed the relationship between the COVID-19 incidence and the SARS-CoV-2 GC quantified in WW at 15 out of 28 WWTPs (overall R2 = 0.36, p < 0.001). At four of the 13 WWTPs where a significant relationship was not found, the GC of SARS-CoV-2 RNA remained below the quantification limit during the whole study period. In the five other WWTPs, the sewer coverage was less than 80% of the total population in the area and thus the COVID-19 cases may have been inhabitants from the areas not covered.Based on the results obtained, WW-based surveillance of SARS-CoV-2 could be used as an indicator for local and national COVID-19 incidence trends. Importantly, the determination of SARS-CoV-2 RNA fragments from WW is a powerful and non-invasive public health surveillance measure, independent of possible changes in the clinical testing strategies or in the willingness of individuals to be tested for COVID-19.

Differentiation of sex chromosomes is thought to have evolved with cessation of recombination and subsequent loss of genes from the degenerated partner (Y and W) of sex chromosomes, which in turn leads to imbalance of gene dosage between sexes. Based on work with traditional model species, theory suggests that unequal gene copy numbers lead to the evolution of mechanisms to counter this imbalance. Dosage compensation, or at least achieving dosage balance in expression of sex-linked genes between sexes, has largely been documented in lineages with male heterogamety (XX/XY sex determination), while ZZ/ZW systems are assumed to be usually associated with the lack of chromosome-wide gene dose regulatory mechanisms. Here, we document that although the pygopodid geckos evolved male heterogamety with a degenerated Y chromosome 32–72 Ma, one species in particular, Burton's legless lizard (Lialis burtonis), does not possess dosage balance in the expression of genes in its X-specific region. We summarize studies on gene dose regulatory mechanisms in animals and conclude that there is in them no significant dichotomy between male and female heterogamety. We speculate that gene dose regulatory mechanisms are likely to be related to the general mechanisms of sex determination instead of type of heterogamety.This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)’.

Grasses are advantageous in the removal of polycyclic aromatic hydrocarbons (PAHs) in soil because of their fibrous root, high tolerance to environmental stress, and low nutritional requirements. In this study, a pot experiment was conducted to test the ability of four grasses to remove PAHs in the soil, and to investigate the corresponding bacterial community shift in the rhizosphere of each. Sudangrass achieved the maximum removal of PAHs at 98% dissipation rate after 20 days. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and next-generation sequencing revealed that sudangrass specially enriched the growth of a known PAHs degrader, Sphingomonadales, regardless of the presence or absence of PAHs in the soil. Moreover, the gene copy numbers of PAHs catabolic genes, PAH-RHDα and nidA, as measured by real time-PCR (RT-PCR) were highest in the soil planted with sudangrass. Overall, this study suggested that sudangrass further enhanced the dissipation of PAHs by enriching Sphingomonadales in its rhizosphere.

Copy number variations (CNVs) are gains and losses of genomic sequence of more than 50 bp between two individuals of a species. Also, CNV is considered to be one of the main elements affecting the phenotypic diversity and evolutionary adaptation of animals. ORMDL sphingolipid biosynthesis regulator 1 (ORMDL1) is a protein-coding gene associated with diseases and development. In our study, the polymorphism of ORMDL1 gene copy numbers in four Chinese sheep breeds (abbreviated CK, HU, STH, and LTH) was detected. In addition, we analyzed the transcriptional expression level of ORMDL1 gene in different tissues of sheep and examined the association of ORMDL1 CNV with growth traits. The statistical analysis revealed that ORMDL1 CNV was remarkably correlated with body height, heart girth, and circumference of cannon bone in HU sheep (P

The effect of low-dose ionizing radiation exposure on leukemia incidence remains poorly understood. Possible dose-response curves for various forms of leukemia are largely based on cohorts of atomic bomb survivors. Animal studies can contribute to an improved understanding of radiation-induced acute myeloid leukemia (rAML) in humans. In male CBA/H mice, incidence of rAML can be described by a two-hit model involving a radiation-induced deletion with Sfpi1 gene copy loss and a point mutation in the remaining Sfpi1 allele. In the present study (historical) mouse data were used and these processes were translated into a mathematical model to study photon-induced low-dose AML incidence in male CBA/H mice following acute exposure. Numerical model solutions for low-dose rAML incidence and diagnosis times could respectively be approximated with a model linear-quadratic in radiation dose and a normal cumulative distribution function. Interestingly, the low-dose incidence was found to be proportional to the modeled number of cells carrying the Sfpi1 deletion present per mouse following exposure. After making only model-derived high-dose rAML estimates available to extrapolate from, the linear-quadratic model could be used to approximate low-dose rAML incidence calculated with our mouse model. The accuracy in estimating low-dose rAML incidence when extrapolating from a linear model using a low-dose effectiveness factor was found to depend on whether a data transformation was used in the curve fitting procedure.

We made the first ever successful effort in India to detect the genetic material of SARS-CoV-2 viruses to understand the capability and application of wastewater-based epidemiology (WBE) surveillance in India. Sampling was carried out on 8 and 27 May 2020 at the Old Pirana Waste Water Treatment Plant (WWTP) at Ahmedabad, Gujarat that receives effluent from Civil Hospital treating COVID-19 patients. All three, i.e. ORF1ab, N and S genes of SARS-CoV-2, were found in the influent with no genes detected in effluent collected on 8 and 27 May 2020. Increase in SARS-CoV-2 genetic loading in the wastewater between 8 and 27 May 2020 samples concurred with corresponding increase in the number of active COVID-19 patients in the city. The number of gene copies was comparable to that reported in untreated wastewaters of Australia, China and Turkey and lower than that of the USA, France and Spain. However, temporal changes in SARS-CoV-2 RNA concentrations need to be substantiated further from the perspectives of daily and short-term changes of SARS-CoV-2 in wastewater through long-term monitoring. The study results SARS-CoV-2 will assist concerned authorities and policymakers to formulate and/or upgrade COVID-19 surveillance to have a more explicit picture of the pandemic curve. While infectivity of SARS-CoV-2 through the excreted viral genetic material in the aquatic environment is still being debated, the presence and detection of genes in wastewater systems makes a strong case for the environmental surveillance of the COVID-19 pandemic., Graphical abstract Unlabelled Image, Highlights • First ever report of the presence of gene of SARS-CoV-2 in the wastewater in India. • CT value is explicitly indicative of the increase of COVID-19 patient in the vicinity. • All three i.e. ORF1ab, N and S genes of SARS-CoV-2 were discerned in the influents. • None of three genes were spotted in the effluent collected on 8 and 27 May 2020. • Increase in the SARS-CoV-2 genetic loading concurred with active COVID-19 patient.

Summary The biological phenotype is affected by the level of gene expression. Here, we provide a step-by-step protocol for precisely titrating and quantitatively observing the target gene expression level in budding yeast by manipulating its copy number in the genome. Using this method, we construct various strains with different gene copy numbers of the cell cycle inhibitor Whi5. This protocol enables stable and inherent control of gene expression at the expected level with fluorescent intensity as the quantitative readout. For complete details on the use and execution of this protocol, please refer to Qu et al. (2019)., Graphical Abstract, Highlights • Precise manipulation of gene copy number in budding yeast. • Stable and inherent control of gene expression level. • Quantitative readout of gene expression level., The biological phenotype is affected by the level of gene expression. Here, we provide a step-by-step protocol for precisely titrating and quantitatively observing the target gene expression level in budding yeast by manipulating its copy number in the genome. Using this method, we construct various strains with different gene copy numbers of the cell cycle inhibitor Whi5. This protocol enables stable and inherent control of gene expression at the expected level with fluorescent intensity as the quantitative readout.

With PCR becoming one of the most important and widely-used diagnostic tools for infectious diseases of poultry, an urgent need has developed for an endogenous internal control (EIC) that monitors the quality and quantity of poultry DNA in test samples. In this study we developed a SYBR-qPCR to target the poultry homolog of the hydroxymethylbilane synthase (HMBS) gene as an EIC for avian species. The avian HMBS-based qPCR was very sensitive, detecting one HMBS gene copy in a 20 µL reaction, and is highly specific for avian species. It amplified DNA from 11 organs and tissues of chickens showing it can be used as an EIC on a large variety of samples. The application of the established EIC on clinically and experimentally infected samples demonstrated that false negativity and result variations could result from samples being collected using different operators, techniques, preservatives, and storage times. The high sensitivity and specificity of the avian HMBS-based qPCR, its ability to quantify DNAs extracted from a wide range of tissues and poultry species along with its usefulness in reducing false negativity in PCR results associated with inadequate sampling and storage degradation makes it an ideal EIC for poultry DNA and RNA PCR diagnostics. The study also highlights the importance of appropriate sampling and storage of samples in ensuring accuracy of molecular diagnostic testing.

Although poultry microbiome discoveries are increasing due to the potential impact on poultry performance, studies examining the poultry respiratory microbiome are challenging because of the low microbial biomass and uniqueness of the avian respiratory tract, making it difficult to sample enough material for microbial analysis. Invasive sampling techniques requiring euthanasia are currently used to increase microbial mass for the analysis, thus making it impossible to sample individual birds longitudinally. In this study, we compared invasive (nasal wash, upper tracheal wash, lower tracheal wash, and lower respiratory lavage) and noninvasive (tracheal and choanal swabs) respiratory sampling techniques in two independent experiments by using 4-wk-old chickens. We first established the experimental baseline of respiratory microbiota by using invasive techniques to enable reasonable comparisons between sampling methods and between experiments. Although noninvasive sampling (live-bird swabs) resulted in lower 16S ribosomal RNA gene copy numbers compared with invasive sampling, live swabs were able to detect the dominant microbes captured by invasive techniques. Nevertheless, swabs from euthanatized birds were more reflective of the microbiota captured through invasive methods than live swab. Furthermore, from two separate experiments, we also demonstrated that respiratory microbiota sampling is highly reproducible, especially in the trachea and lower respiratory tract. Our study provides new insights and perspectives on decision making when sampling and studying poultry respiratory microbiota.

Specific quantification of root-colonizing arbuscular mycorrhizal fungi (AMF) by quantitative real-time PCR is a high-throughput technique, most suitable for determining abundances of AMF species or isolates in previously characterized experimental systems. The principal steps are the choice and validation of an appropriate assay to specifically amplify a gene fragment of the target AMF, preparation of templates from root samples, and quantification of the fungal gene copy numbers in these templates. The use of a suitable assay is crucial for a correct data collection but also highly specific for each experimental system and is therefore covered by general recommendations. Subsequently, specific steps are described for the validation of the assay using a standard dilution series, the determination of appropriate dilutions of DNA extracts from roots, and the quantification of the gene copy numbers in samples including calculations.

Gene augmentation therapy entails the intracellular introduction of a healthy gene copy to modify the expression of a gene product. The fundamental cause of a disease is hereby treated as individual cell function is restored. In 2017, the US Food and Drug Administration approved a gene therapy product indicated for the treatment of patients with Leber congenital amaurosis (LCA). At present, gene therapy is being developed for various other inherited retinal diseases (IRDs) and multiple clinical trials are currently in progress. This chapter contains an overview of the current clinical trials involving gene augmentation therapy for IRDs. In addition, the current knowledge about ocular gene therapy is being described.

The relationship between serum concentration of complement C4 ([C4]) and C4 gene copy number (GCN) was investigated in 56 systemic lupus erythematosus (SLE) patients and 33 age and sex-matched controls in a Western Australian population. C4A and C4B gene copy numbers (C4A & B GCN) together with the presence or absence of the ≈6.4-kb human endogenous retroviral element type K (hereafter HERV-K) in intron 9 were estimated by two TaqMan™ real-time PCR (RT-PCR) assays that measured total C4 and HERV-K GCNs, respectively. There was good correlation between the two methods; however, the HERV-K GCN method showed a positive bias (≈6%) relative to the C4A & B total GCN. Despite individual variation, excellent correlation between total C4 GCN and mean [C4] per GCN was observed for both the SLE and control cohorts ( R2 = 88% and R2 = 99%, respectively). It was noted that serum [C4] was significantly lower in the SLE patients than the controls ( p = 0.006) despite there being no difference between C4A and C4B GCN in both cohorts. The data therefore confirm previous reports that the C4A genes are preferentially associated with the presence of the HERV-K insertion relative to C4B genes and does not support the hypothesis that low [C4] in SLE is explained by low C4A GCNs. There was no evidence also that the presence of the HERV-K insertion in C4 genes influenced [C4]. This study supports the view that low [C4] in SLE patients is due to consumption rather than deficient synthesis related to lower C4A & B GCN.

Neutral evolution is the default process of genomic changes. This is because our world is finite, and the randomness, indispensable for neutral evolution, is important when we consider the history of a finite world. The random nature of DNA propagation is discussed using branching process, coalescent process, Markov process, and diffusion process. Expected evolutionary patterns under neutrality are then discussed on fixation probability, rate of evolution, and amount of DNA variation kept in population. We then discuss various features of neutral evolution starting from evolutionary rates, synonymous and nonsynonymous substitutions, junk DNA, and pseudogenes.

AIMS: The overarching aims of this study were to determine if microbes found in wastewater treatment plants (WWTPs) carry silver resistance genes and ascertain whether a relationship exists between silver loading and sil silver resistance gene copy numbers. METHODS AND RESULTS: Activated sludge samples were collected from 17 WWTPs across the United States and characterized for chemical oxygen demand (COD) and total silver concentration. Gene copy numbers were quantified using qPCR for four common silver resistance genes (silC, silP, silR and silS). Spearman correlation analyses were performed to examine associations between three WWTP characteristics (i.e. size, COD and total silver concentration) and sil gene copy numbers. sil genes were found in all activated sludge samples, however the quantity of gene copy numbers was inconsistent. Of the sil genes screened, silS was the most abundant, followed by silR. Gene copy numbers for silC and silP were generally similar and overall lower than silS and silR. CONCLUSIONS: Neither individual or combined sil gene copy numbers correlated significantly with total silver concentration, suggesting either the presence of alternate mechanisms of silver resistance or that a critical silver concentration threshold within WWTPs was not met that would require specific resistance against silver. SIGNIFICANCE AND IMPACT OF THIS STUDY: To the authors' knowledge, this is one of the first studies to measure the prevalence of sil genes in engineered systems. Results from this study suggest that at least four silver resistance genes are commonly found within WWTP‐activated sludge, however it is unclear if these genes are being expressed under current total silver loadings.

Increasing growth rate across bacteria strengthens selection for faster translation, concomitantly increasing the total number of tRNA genes and codon usage bias (CUB: enrichment of specific synonymous codons in highly expressed genes). Typically, enriched codons are translated by tRNAs with higher gene copy numbers (GCN). A model of tRNA–CUB coevolution based on fast growth-associated selection on translational speed recapitulates these patterns. A key untested implication of the coevolution model is that translational selection should favor higher tRNA GCN for more frequently used amino acids, potentially weakening the effect of growth-associated selection on CUB. Surprisingly, we find that CUB saturates with increasing growth rate across γ-proteobacteria, even as the number of tRNA genes continues to increase. As predicted, amino acid-specific tRNA GCN is positively correlated with the usage of corresponding amino acids, but there is no correlation between growth rate associated changes in CUB and amino acid usage. Instead, we find that some amino acids—cysteine and those in the NNA/G codon family—show weak CUB that does not increase with growth rate, despite large variation in the corresponding tRNA GCN. We suggest that amino acid-specific variation in CUB is not explained by tRNA GCN because GCN does not influence the difference between translation times of synonymous codons as expected. Thus, selection on translational speed alone cannot fully explain quantitative variation in overall or amino acid-specific CUB, suggesting a significant role for other functional constraints and amino acid-specific codon features.

SARS-CoV-2 wastewater epidemiology suffers from uncertainties concerning sample storage. We show the effect of storage of wastewater on the detectable SARS-CoV-2 load. Storage at 4 °C up to 9 days had no significant effect, while storage at −20 °C led to a significant reduction in gene copy numbers.HighlightsRaw wastewater samples can be stored up to 9 days at 4°C for SARS-CoV-2 wastewater surveillanceFreezing of wastewater sample dramatically decreases RT-qPCR signal of SARS-CoV-2 from wastewater.

The effects of functional membrane covering (FMC) on nitrogen transformation and related functional genes during aerobic composting were investigated by performing a comparable experiment. The FMC increased the pile temperature, promoted compost maturity, and decreased nitrogen loss. The FMC reduced NH3 and N2O emissions by 7.34% and 26.27%, respectively. The water film and the micro-positive pressure environment under the membrane effectively prevented NH3 escaping. The FMC up-regulated the amoA gene copy number (promoting NH3/NH4+ oxidation). The reduction of N2O emission by the FMC was mainly related to denitrifying genes (nirK, nirS, and nosZ). The FMC down-regulated the nirK and nirS gene copy numbers, but up-regulated the nosZ gene copy number. Pearson correlation analysis indicated that the functional membrane characteristics and differences between the composting pile environments caused by the FMC significantly affected the nitrogen forms and the related functional genes. The FMC strongly decreased nitrogen emissions and therefore conserved nitrogen.

Quantitative real-time PCR (qPCR) analysis for accurate quantification of targeted microbial genes is compromised by the presence of co-extracted inhibitors from soil samples. Dilution of DNA extracts is a commonly-used method to reduce levels of inhibition. However, the applications of dilution method are mostly empirical, and need to be further elaborated. Here, we propose a dilution model to re-evaluate dilution as a method to eliminate qPCR inhibition. We found that DNA extracts without dilution or with a minor dilution (e.g., 10-fold) resulted in qPCR inhibition for most of studied soils. However, excessive dilution (e.g., 200- or 400-fold) caused an overestimation of the quantified gene copy numbers. Only under a moderate dilution range could qPCR inhibition be efficiently eliminated, which has been well captured by our proposed dilution model. The pre-testing of qPCR inhibition for determining the appropriate dilution range for extracted DNA samples aids accurate quantification of nucleic acids in soils.

Glyphosate-resistant (GR) kochia is an increasing concern for growers across the U.S. Great Plains and Canadian prairies. Integrated strategies to mitigate resistance will require an improved understanding of the seed germination dynamics of GR kochia populations. Experiments were conducted to characterize the germination of GR vs. glyphosate-susceptible (GS) kochia populations under different constant (5 to 35 C) and alternating (5/10 to 30/35 C) temperatures. Seven GR and two GS populations were collected from wheat–fallow fields in northern Montana. Selected lines of GR and GS were obtained after three generations of recurrent group selection in the greenhouse. The GR-selected lines had 4.1 to 10.8 averageEPSPScopies compared with a singleEPSPSgene copy for the GS selected lines. Four out of seven GR selected lines had lower final germination (dparameter) and took more time to complete 50% cumulative germination (I50values) under all constant and alternating temperatures, compared with the GS selected lines. Those GR selected lines also had a delayed germination initiation (I10values), particularly at lower temperatures (5 to 10 C constant or 5/10 C alternating). In contrast, the final germination (d) of the other three GR selected lines did not differ from GS lines at a majority of temperatures tested. The I50values of those GR lines were also comparable to GS lines under a majority of the temperatures. There was no significant correlation of observed percent cumulative germination andEPSPSgene copy number of selected kochia lines. The temperature-dependent dormancy and altered germination characteristics of the four GR kochia lines reflect the common selection of resistance and avoidance (glyphosate or other preseeding treatments) mechanisms. This is most likely attributed to long-term, intensive cropping practices and less diverse weed control methods, rather than a fitness cost or pleiotropic effect of multiple copies of theEPSPSgene.

Most higher eukaryotes are diploid organisms. Most genes are expressed from both chromosomal copies, but there are several important exceptions, in particular, the X-chromosome in mammals, imprinted genes, or genes of the immune system. In some cases, it was shown that the functioning of only one gene copy is determined by the interaction between its sense and antisense transcripts. In this work, a minimal model of strictly monoallelic expression is proposed and analyzed.

There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the ΔΔCt formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies.

Nontuberculous mycobacteria (NTM) can carry two or more 16S rRNA gene copies that are, in some instances, non-identical. In this study, we used a combined cloning and sequencing approach to analyze 16S rRNA gene sequences of six NTM species, Mycobacterium cosmeticum, M. pallens, M. hodleri, M. crocinum, M. flavescens, and M. xenopi. Our approach facilitated the identification of two distinct gene copies in each species. The two M. cosmeticum genes had a single nucleotide difference, whereas two nucleotide polymorphisms were identified in M. hodleri, M. flavescens, and M. xenopi. M. pallens had a difference in four nucleotides and M. crocinum ? in 23 nucleotides. Thus, we showed that the six NTM species possess at least two non-identical 16S rRNA gene copies. The full-length sequences of the intraspecies 16S rRNA variants will facilitate NTM identification and sequence analysis of specimens or other samples., Journal of Microbiological Methods, 155, pp.34-36; 2018

The 16S rRNA gene is the golden standard target of sequencing to uncover the composition of bacterial communities but the presence of multiple copies of the gene makes gene copy normalization (GCN) inevitable. Even though GCN resulted in abundances closer to the metagenome, it should be validated by communities with known composition as both amplicon and shotgun sequencing are prone to methodological biases. Here we compared the composition of three mock communities to the composition derived from 16S sequencing without and with GCN. In all of them, the 16S composition was different from the mock community and GCN improved the picture only in the community with the lowest Shannon diversity. Albeit with low abundance, half of the identified genera were not present in the mock communities. Our approach provides empirical evidence to the methodological biases introduced by sequencing that was only counteracted by GCN in the case of low α-diversity, potentially due to the small number of bacterial taxa with known gene copy numbers. We thus cannot recommend the use of GCN moving forward and it is questionable whether a complete catalogue of 16S rRNA copy numbers can outweigh the methodological biases of sequencing.

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications) in addition to sequence variants. While guidelines for the reporting of sequence variants are available, guidance with respect to reporting copy number gains from gene-panel NGS data is limited. Here, we report on Dutch consensus recommendations obtained in the context of the national Predictive Analysis for THerapy (PATH) project, which aims to optimize and harmonize routine diagnostics in molecular pathology. We briefly discuss two common approaches to detect gene copy number gains from NGS data, i.e., the relative coverage and B-allele frequencies. In addition, we provide recommendations for reporting gene copy gains for clinical purposes. In addition to general QC metrics associated with NGS in routine diagnostics, it is recommended to include clinically relevant quantitative parameters of copy number gains in the clinical report, such as (i) relative coverage and estimated copy numbers in neoplastic cells, (ii) statistical scores to show significance (e.g., z-scores), and (iii) the sensitivity of the assay and restrictions of NGS-based detection of copy number gains. Collectively, this information can guide clinical and analytical decisions such as the reliable detection of high-level gene amplifications and the requirement for additional in situ assays in case of borderline results or limited sensitivity. Electronic supplementary material The online version of this article (10.1007/s00428-019-02555-3) contains supplementary material, which is available to authorized users.

A ranked tree topology is a tree topology with a temporal ordering of its coalescence events. Under the multispecies coalescent model, we consider ranked gene tree topologies realized along the branches of ranked species trees, where one gene copy is sampled for each species. Previous results have demonstrated that for almost all ranked species tree topologies with at least five species, there exists a set of branch lengths such that the maximally probable ranked gene tree topologies-those generated with the highest probability under the model-do not match the species tree ranked topology. Here, we focus on the agreement of a ranked species tree with its maximally probable ranked gene tree topologies in terms of their unranked topology, that is, disregarding the ordering of the coalescence events. We show that although the set of maximally probable ranked gene tree topologies for a ranked species tree can contain ranked trees with different unranked topologies, at least one of these maximal ranked gene tree topologies must have the same unranked topology as the species tree. Our results contribute to the study of the relationships between gene trees and species trees.

This study aimed to quantity total numbers of bacteria, fungi and archaea in different types of commercial liquid anaerobic digestates, and to identify common patterns in their microbial numbers post-digestion and possible implications of their use as biofertiliser. Relationships between microbial numbers and physical-chemical traits of the digestates were also investigated. Quantification was performed using culturable and molecular (quantitative PCR) approaches. Bacterial and fungal CFUs ranged up to five orders of magnitude (105 to 1010; 0 to 105 g-1 DW, respectively) between different types of anaerobic digestates. Bacterial, archaeal and fungal gene copy numbers (GCN) varied by two orders of magnitude (108 to 1010; 107 to 109; 104 to 106 g-1 DW, respectively) between digestates. All microbial variables analysed showed significant differences between the different types of anaerobic digestate investigated (p 4 CFU g-1 DW) were much lower than for bacteria (2.23 × 109 CFU g-1 DW). Gene copy numbers were highest for bacteria (16S) (1.09 × 1010 g-1 DW), followed by archaea (16S) (5.87 × 108 g-1 DW), and fungi (18S) (1.77 × 106 g-1 DW). Liquid anaerobic digestates were predominantly dominated by bacteria, followed by archaeal and fungal populations. At 50% similarity level, the microbial profiles of the eleven anaerobic digestates tested separated into just two groups, indicating a broad relative degree of similarity in terms of microbial numbers. Higher bacterial (16S) GCN was associated with low OM and C/N ratio in digestates.

Infectious disease-causing pathogens can be transmitted via contact with contaminated environmental surfaces (i.e., fomite transmission). However, the physical mechanism of fomite transmission remains unclear because of a lack of suitable surrogate tracers. In this study, the non-toxic, non-human-origin and gram-positive bacterium Lactobacillus delbrueckii subsp. Bulgaricus was selected as a tracer, and its applicability for studying bacterial contamination spread on environmental surfaces was investigated in an office setting. After bacterial introduction by a designated carrier, surface contamination levels indicated by the L. bulgaricus proline iminopeptidase (pepIP) gene copy numbers were quantified using an optimized qPCR assay. This qPCR assay exhibited a high sensitivity (55 copies per reaction mixture and 1585 copies per surface sample), a wide dynamic range (7 orders of magnitude) and a high specificity for pepIP gene in environmental surface samples. Three independent, natural and unsupervised field experiments involving human participants confirmed the applicability of L. bulgaricus as a tracer in field studies and the pepIP gene as a unique marker of externally introduced contamination. Experimentally, contamination was distributed nearly equally over public surfaces, the carrier's seat location and the other seat locations in the office setting. An analysis by seat location revealed that the contamination level was attenuated as the physical distance from the carrier's seat location increased. Volunteers' faces were ubiquitously contaminated, suggesting a potentially high risk of fomite exposure via mucous membranes. In conclusion, L. bulgaricus can feasibly be used as a tracer organism in future large-scale field studies of bacterial contamination spread on environmental surfaces.

Gut-stimulated denitrifiers are responsible for the in vivo emission of N 2 O by earthworms, and contribute to the N 2 O that is emitted from certain terrestrial ecosystems. To evaluate the separate effect of gut-stimulated denitrifiers on N 2 O emissions, earthworms or their casts were inoculated into sterilized and unsterilized soil, respectively, and compared with control (soil without inoculation) in this study, to find out if there was a difference in N 2 O emissions during the 15 days incubation period. Compared with the control, earthworm inoculation significantly increased N 2 O flux and relative densities of nirS and narG gene copy numbers both in sterilized and unsterilized soil, while no significant difference was found when we transited and recolonized those gut-stimulated denitrifiers into soil artificially. In contrast, cast inoculation did not change CO 2 emission in the unsterilized soil, but significant increased cumulative CO 2 emission in the sterilized soil. Our data suggest that the effect of gut-stimulated denitrifiers on soil N 2 O produce was strongly diminished when separated from earthworm activity.

About 25 golf courses in Ontario, Canada have environmental compliance approvals to use reclaimed water for irrigation, where disinfection is confirmed through E. coli limits. A previous study at five Ontario municipal wastewater treatment plants (WWTPs) confirmed that enteric viruses are less susceptible to disinfection than E. coli, when plants provided conventional (secondary or tertiary) treatment and routine (chlorine or UV) disinfection. Here we query whether these four treatment-disinfection scenarios plus 60-day lagoon storage of disinfected effluent would be sufficient to reduce norovirus genogroups I and II (NoV GI and GII) risk of infection to tolerable levels for a golfer who incidentally ingests NoV after handling wet golf balls at a golf course irrigated with reclaimed water. We used our RT-qPCR NoV enumeration datasets from the four treatment-disinfection scenarios above and modeled detected and non-detected NoV by Bayesian inference in ‘R’. Monte Carlo simulation included pre-disinfection NoV GI and GII gene copy densities; Ontario WWTP-derived chlorine and UV log10 reductions; literature-derived effluent storage decay parameters and golfer ingested volumes, followed by six different NoV dose-response (DR) models. Quantitative Microbial Risk Assessment (QMRA) results suggest that there is an unacceptable NoV infection risk when using the conservative assumption that all detected NoV particles (RT-qPCR gene copies) are infectious, in both aggregated or disaggregated form. However, after adjusting for PCR target sequences and for infectiousness using data from recently published studies on cultivation of human NoV in human intestinal enteroids, we noted a significant reduction of infection risk. However, this less conservative (i.e., less protective) assumption for water reuse applications such as golf course irrigation may not be corroborated until human NoV are efficiently and routinely grown in cell cultures. In addition, further studies on drivers of NoV risk estimation by DR models are needed, e.g., the extent of NoV particle aggregation resulting from wastewater treatment, as well as the role of immunity. Meantime, regulatory agencies could consider more stringent treatment-disinfection requirements that target enteric viruses rather than E. coli and testing of actual reclaimed irrigation waters.

e18029 Background: Numerous studies on cervical cancer confirm an important role of specific genomic changes in the onset and development of cervical intraepithelial neoplasia and their effect on the progression of cervical cancer. Solid tumors are characterized by genomic changes leading to a change in the DNA sequence copy number. The purpose of the study was to reveal changes in the relative copy number of the ESR1, ESR2, GPER1, STS, SULT1A1, SULT1E1, CYP1A1, CYP1A2 genes responsible for the reception and metabolism of estrogens in cervical tissues in endophytic and exophytic patterns of tumor growth in order to find predictive markers of malignancy. Methods: The study included 40 patients aged 28-65 years with cervical cancer of endophytic (n = 20) and exophytic (n = 20) growth patterns. Eligibility criteria included a morphologically confirmed cervical squamous cell cancer T1b-2aN0M0, stage I-II. The Thermo Scientific GeneJET FFPE DNA Purification Kit was used for the DNA extraction from FFPE blocks of tumor and healthy tissues. DNA concentrations were measured on the Qubit 2.0 fluorimeter (Invitrogen, USA) using the Quant-iT dsDNA High-Sensitivity (HS) Assay Kit (Invitrogen, USA). Results: The relative copy number of the GPER1, SULT1A1, CYP1A1 genes in tumor samples increased (p < 0.05) compared with normal tissues in the total sample of patients diagnosed with cervical cancer. In contrast to the total sample, an increase in the SULT1A1 gene dosage did not reach a statistically significant level in any group (p = 0.242 and p = 0.157); the copy number of the GPER1 locus significantly increased only in the group with the endophytic growth pattern (p = 0.040), as well as the CYP1A2 gene dosage (p = 0.025). Patients of 36-55 years with endophytic tumors showed a statistically significant (p < 0.05) increase in the GPER1 and CYP1A1 gene copy numbers with the rates of 41.7% and 66.7%, respectively, as well as an increased amplification of the CYP1A2 gene in 41.67% of patients. In women of 56-75 years with endophytic tumors, an increase in the copy numbers of the ESR2, GPER1, SULT1A1 genes was observed with a frequency of 50%, 100% and 75%, respectively. Patients aged 20-35 and 36-55 years with exophytic tumors showed a statistically significant (p < 0.05) increase in the CYP1A1 gene copy numbers in 33.33% and 45.45%, respectively. Conclusions: The results suggest the use of the GPER1, SULT1A1 and CYP1A1 gene copy numbers as biomarkers of cervical tumors.

Hot water premise plumbing has emerged as a critical nexus of energy, water, and public health. The composition of hot water microbiomes is of special interest given daily human exposure to resident flora, especially opportunistic pathogens (OPs), which rely on complex microbial ecological interactions for their proliferation. Here, we applied shotgun metagenomic sequencing to characterize taxonomic and functional shifts in microbiomes as a function of water heater temperature setting, stagnation in distal pipes, and associated shifts in water chemistry. A cross-section of samples from controlled, replicated, pilot-scale hot water plumbing rigs representing different temperature settings (39, 42, and 51 degrees C), stagnation periods (8 h vs. 7 days), and time-points, were analyzed. Temperature setting exhibited an overarching impact on taxonomic and functional gene composition. Further, distinct taxa were selectively enriched by specific temperature settings (e.g., Legionella at 39 degrees C vs. Deinococcus at 51 degrees C), while relative abundances of genes encoding corresponding cellular functions were highly consistent with expectations based on the taxa driving these shifts. Stagnation in distal taps diminished taxonomic and functional differences induced by heating the cold influent water to hot water in recirculating line. In distal taps relative to recirculating hot water, reads annotated as being involved in metabolism and growth decreased, while annotations corresponding to stress response (e.g., virulence disease and defense, and specifically antibiotic resistance) increased. Reads corresponding to OPs were readily identified by metagenomic analysis, with L. pneumophlla reads in particular correlating remarkably well with gene copy numbers measured by quantitative polymerase chain reaction. Positive correlations between L. pneumophila reads and those of known protozoan hosts were also identified. Elevated proportions of genes encoding metal resistance and hydrogen metabolism were noted, which was consistent with elevated corrosion-induced metal concentrations and hydrogen generation. This study provided new insights into real-world factors influencing taxonomic and functional compositions of hot water microbiomes. Here metagenomics is demonstrated as an effective tool for screening for potential presence, and even quantities, of pathogens, while also providing diagnostic capabilities for assessing functional responses of microbiomes to various operational conditions. These findings can aid in informing future monitoring and intentional control of hot water microbiomes.

WOS: 000434056100015, PubMed ID: 29432640, BackgroundThis study compares diamond burs and curettes by clinical, microbiological, biochemical and scanning electron microscopic parameters and treatment time data in the non-surgical periodontal treatment of patients with chronic periodontitis. MethodsTwo quadrants of each of the 12 patients received root planing with diamond burs, whereas the other two quadrants were treated with curettes. Clinical periodontal measurements were recorded at baseline and then 1, 3 and 6months after completion of non-surgical periodontal treatment. Subgingival plaque and gingival crevicular fluid samples were obtained at baseline and 1-month control. Twenty-one hopeless teeth received root planing with diamond burs or curettes or no treatment and then extracted for microscopic evaluations. ResultsClinical periodontal parameters improved similarly with both treatment modalities. Microbiological analyses revealed similar findings for the bacterial load (16S gene copy numbers) and ratio of each bacterium to the total bacterial count at baseline and 1-month control. Cytokine levels in the gingival crevicular fluid samples exhibited differences between the two treatments. Scanning electron microscopic analyses indicated that diamond burs were better in terms of calculus removal and loss of tooth substance indices but roughness index values were better for curettes. ConclusionsDiamond burs provide findings comparable with curettes in root planing., Ege University Research FoundationEge University [2013-DIS-008]; Department of Periodontology, School of Dentistry, Ege University, Izmir, Turkey; Oral Sciences Research Group, Dental Hospital and School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, UK, The work was supported solely by the Department of Periodontology, School of Dentistry, Ege University, Izmir, Turkey and Oral Sciences Research Group, Dental Hospital and School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, UK. This study was supported by a grant from the Ege University Research Foundation (Project No: 2013-DIS-008).

In this era of precision medicine, molecular testing of tumor specimens has become indispensible in order to seek opportunities for optimized and personalized treatment in every cancer patient. Since most pivotal trials leading to the approval of targeted drugs rely on tissue specimens for predictive marker testing, biomarker testing on tissue biopsy specimens has long been considered the gold standard. In the real diagnostic world, however, malignant tumors are often diagnosed by cytology alone. This necessitates translating protocols for biomarker testing for the use in cytology, which comes with advantages and challenges. There is a general consensus that the quality of DNA obtained from ethanol-based cytological specimens is excellent allowing for highly accurate mutation analysis. Enrichment of tumor cells by laser capture microdissection is a powerful and robust method to rescue tumor cytological specimens with a low tumor cell proportion for DNA-based analysis. The fact that cells on smears, cytospins, or liquid-based cytology (LBC) preparations are intact makes them also an ideal substrate for the analysis of chromosome and gene copy numbers or gene rearrangements by fluorescence in situ hybridization. In contrast, the great variability of preparation, fixation, and staining methods in cytology is a hurdle toward standardized protocols for predictive immunohistochemistry. This can be partly overcome by cell blocks that can be processed like formalin-fixed and paraffin-embedded tissue specimens for biomarker testing. Although there is need for more standardization in protocols of molecular testing on cytological specimens, the utility of cytology for biomarker testing is now undisputed.

Practical difficulties of the traditional adenovirus infectivity assay such as intensive labor requirements and longer turnaround period limit the direct use of adenovirus as a testing microorganism for systematic, comprehensive disinfection studies. In this study, we attempted to validate the applicability of integrated cell culture quantitative PCR (ICC-qPCR) as an alternative to the traditional cell culture method with human adenovirus type 2 (HAdV2) in a low-pressure UV disinfection study and to further optimize the procedures of ICC-qPCR for 24-well plate format. The relatively high stability of the hexon gene of HAdV2 was observed after exposure to UV radiation, resulting in a maximum gene copy reduction of 0.5 log10 at 280 mJ cm(-2). Two-day post-inoculation incubation period and a maximum spiking level of 10(5) MPN mL(-1) were selected as optimum conditions of ICC-qPCR with the tested HAdV2. An approximate 1:1 correlation of virus quantities by the traditional and ICC-qPCR cell culture based methods suggested that ICC-qPCR is a satisfactory alternative for practical application in HAdV2 disinfection studies. ICC-qPCR results, coupled with a first-order kinetic model (i.e., the inactivation rate constant of 0.0232 cm(2) mJ(-1)), showed that an UV dose of 172 mJ cm(-2) achieved a 4-log inactivation credit for HAdV2. This estimate is comparable to other studies with HAdV2 and other adenovirus respiratory types. The newly optimized ICC-qPCR shows much promise for further study on its applicability of other slow replicating viruses in disinfection studies.

In this study, qPCR was used to quantify opportunistic premise plumbing pathogens (OPPPs) and free-living amoebae in 11 tap water samples collected over four seasons from a city in northern China. Results demonstrated that the average numbers of gene copies of Legionella spp. and Mycobacterium spp. were significantly higher than those of Aeromonas spp. (p 0.05). Legionella spp. and Mycobacterium spp. were 100% (44/44) positively detected while P. aeruginosa and Aeromonas spp. were 79.54% (35/44) and 77.27% (34/44) positively detected. Legionella pneumophila was only detected in 4 samples (4/44), demonstrating its occasional occurrence. No Mycobacterium avium or Naegleria fowleri was detected in any of the samples. The average gene copy numbers of target OPPPs were the highest in summer, suggesting seasonal prevalence of OPPPs. Average gene copy numbers of OPPPs in the taps of low-use-frequency were higher than in taps of high-use-frequency, but the difference was not significant for some OPPPs (p 0.05). Moderate negative correlations between the chlorine concentration and the gene copy numbers of OPPPs were observed by Spearman analysis (r

Carboxylesterase 1 (CES1) is implicated in the metabolism of several commonly used drugs and other xenobiotics. The gene encoding this enzyme, CES1, is duplicated in some individuals. The original gene copy is called CES1A1. The duplicated version, CES1A2, is a hybrid of CES1A1 and the CES1-related pseudogene, CES1P1. Variants of CES1A2 with a weak and a strong promoter, respectively, have been reported. In addition, there are chimeric subtypes of CES1A1 that contain a segment of CES1P1. Collectively, this represents challenges to the genotyping of CES1 that previous procedures have had difficulties in solving, frequently leading to loss of specificity and inaccurate genotyping. Here, we report a novel and specific procedure that can selectively amplify CES1A1 and CES1A2 and accurately determine their variants. This procedure may be useful for personalization of treatments with drugs metabolized by CES1.