Development of Presumptive Sialadenosis Following a Chronic Oropharyngeal Stick Injury in a Dog.

A 3 yr old female spayed Labrador retriever was referred for the treatment of a chronic oropharyngeal stick injury. After computed tomography scan evaluation, the cervical area was explored surgically and a right-sided cervical abscess that contained a wooden stick was identified adjacent to the vagosympathetic trunk and carotid artery. The ipsilateral mandibular salivary gland was resected concurrently given its abnormal appearance, and histology confirmed inflammation and necrosis of the gland, which was suspected to be due to direct trauma from the foreign body. The clinical signs initially improved but then recurred, and a follow-up computed tomography scan was suggestive of sialadenosis or sialadenitis in the right parotid, zygomatic, and molar salivary glands. A presumptive diagnosis of sialadenosis was made and a course of phenobarbital was initiated. The clinical signs resolved completely within a few days, and there was no recurrence several months after termination of the phenobarbital treatment. This is the first case report of presumptive sialadenosis in a dog as a suspected complication of an oropharyngeal stick injury. Informed consent was obtained from the owner of the dog and the patient was managed according to contemporary standards of care.

Shedding light on AT1G29480 of Arabidopsis thaliana-An enigmatic locus restricted to Brassicacean genomes.

A key feature of C4 Kranz anatomy is the presence of an enlarged, photosynthetically highly active bundle sheath whose cells contain large numbers of chloroplasts. With the aim to identify novel candidate regulators of C4 bundle sheath development, we performed an activation tagging screen with Arabidopsis thaliana. The reporter gene used encoded a chloroplast-targeted GFP protein preferentially expressed in the bundle sheath, and the promoter of the C4 phosphoenolpyruvate carboxylase gene from Flaveria trinervia served as activation tag because of its activity in all chlorenchymatous tissues of A. thaliana. Primary mutants were selected based on their GFP signal intensity, and one stable mutant named kb-1 with a significant increase in GFP fluorescence intensity was obtained. Despite the increased GFP signal, kb-1 showed no alterations to bundle sheath anatomy. The causal locus, AT1G29480, is specific to the Brassicaceae with its second exon being conserved. Overexpression and reconstitution studies confirmed that AT1G29480, and specifically its second exon, were sufficient for the enhanced GFP phenotype, which was not dependent on translation of the locus or its parts into protein. We conclude, therefore, that the AT1G29480 locus enhances the GFP reporter gene activity via an RNA-based mechanism.

Comparison of meiotic transcriptomes of three maize inbreds with different origins reveals differences in cell cycle and recombination.

BACKGROUND: Cellular events during meiosis can differ between inbred lines in maize. Substantial differences in the average numbers of chiasmata and double-strand breaks (DSBs) per meiotic cell have been documented among diverse inbred lines of maize: CML228, a tropical maize inbred line, B73 and Mo17, temperate maize lines. To determine if gene expression might explain these observed differences, an RNA-Seq experiment was performed on CML228 male meiocytes which was compared to B73 and Mo17 male meiocytes, where plants were grown in the same controlled environment. RESULTS: We found that a few DSB-repair/meiotic genes which promote class I crossovers (COs) and the Zyp1 gene which limits newly formed class I COs were up-regulated, whereas Mus81 homolog 2 which promotes class II COs was down-regulated in CML228. Although we did not find enriched gene ontology (GO) categories directly related to meiosis, we found that GO categories in membrane, localization, proteolysis, energy processes were up-regulated in CML228, while chromatin remodeling, epigenetic regulation, and cell cycle related processes including meiosis related cell cycle processes were down-regulated in CML228. The degree of similarity in expression patterns between the three maize lines reflect their genetic relatedness: B73 and Mo17 had similar meiotic expressions and CML228 had a more distinct expression profile. CONCLUSIONS: We found that meiotic related genes were mostly conserved among the three maize inbreds except for a few DSB-repair/meiotic genes. The findings that the molecular players in limiting class I CO formation (once CO assurance is achieved) were up-regulated and those involved in promoting class II CO formation were down-regulated in CML228 agree with the lower chiasmata number observed in CML228 previously. In addition, epigenetics such as chromatin remodeling and histone modification might play a role. Transport and energy-related processes was up-regulated and Cyclin13 was down-regulated in CML228. The direction of gene expression of these processes agree with that previously found in meiotic tissues compared with vegetative tissues. In summary, we used different natural maize inbred lines from different climatic conditions and have shown their differences in expression landscape in male meiocytes.

Profiling m6A RNA Modifications in Low Amounts of Plant Cells Using Maize Meiocytes.

RNA modifications can influence gene expression via multiple aspects such as RNA stability and alternative splicing. The most prominent RNA modification is m6A (N6-methyladenosine). Its profiling from low starting amounts of <100 cells is challenging. We describe here a complete workflow from cell isolation to data analysis that is based on using an RNA CUT&RUN-supported m6A-RIP (RNA immunoprecipitation) procedure and a subsequent adaptor-tagging library synthesis. Male meiocytes isolated from maize anthers were used as a test system to establish the protocol.

UV-B Irradiation Results in Inhibition of Hypocotyl Elongation, Cell Cycle Arrest, and Decreased Endoreduplication Mediated by miR5642.

UV-B as a component of natural solar radiation can induce damage and morphological development in plants. The UV-B response from germination and early development in seedlings is still largely unknown, with most studies focused on older, light-exposed seedlings. We used fluence response curves measuring hypocotyl length after UV-B exposure coupled with RNA-seq and sRNA-seq evaluation of the early seedling response in the model organism Arabidopsis thaliana. We identified miR5642 as a potential novel key regulator of UV-B responses. miR5642 is a noncanonical miRNA predicted to target previously known and unknown components involved in hypocotyl growth inhibition. These include (i) SMAX1, a signal transmitter for seedling germination and growth; (ii) ZAT1, an uncharacterized transcription factor; and (iii) membrane pores and transporters (VHA-E1, VHA-E3, EPSIN-LIKE and PIP1.4) implicated in cell elongation. In addition, HY5 and HYH, two homologous and redundant transcription factors involved in seedling photomorphogenesis, may interact with these newly identified components. Interestingly, UV-B-induced DNA photodimer formation seems to be the direct trigger leading to inhibition of hypocotyl growth through a combination of cellular decisions including cell cycle arrest, reduced endoreduplication and reduced cell elongation, and this inhibition appears to be modulated by miR5642 target genes.

IER2-induced senescence drives melanoma invasion through osteopontin.

Expression of the immediate-early response gene IER2 has been associated with the progression of several types of cancer, but its functional role is poorly understood. We found that increased IER2 expression in human melanoma is associated with shorter overall survival, and subsequently investigated the mechanisms through which IER2 exerts this effect. In experimental melanoma models, sustained expression of IER2 induced senescence in a subset of melanoma cells in a p53/MAPK/AKT-dependent manner. The senescent cells produced a characteristic secretome that included high levels of the extracellular phosphoglycoprotein osteopontin. Nuclear localization of the IER2 protein was critical for both the induction of senescence and osteopontin secretion. Osteopontin secreted by IER2-expressing senescent cells strongly stimulated the migration and invasion of non-senescent melanoma cells. Consistently, we observed coordinate expression of IER2, p53/p21, and osteopontin in primary human melanomas and metastases, highlighting the pathophysiological relevance of IER2-mediated senescence in melanoma progression. Together, our study reveals that sustained IER2 expression drives melanoma invasion and progression through stimulating osteopontin secretion via the stochastic induction of senescence.

Oxygen, secreted proteins and small RNAs: mobile elements that govern anther development.

Correct anther development is essential for male fertility and subsequently agricultural yield. Defects in anther development range from the early stage of stamen formation until the late stage of tapetum degeneration. In particular, the specification of the four distinct somatic layers and the inner sporogenous cells need perfect orchestration relying on precise cell-cell communication. Up to now, several signals, which coordinate the anther´s developmental program, have been identified. Among the known signals are phytohormones, environmental conditions sensed via glutaredoxins, several receptor-like kinases triggered by ligands like MAC1, and small RNAs such as miRNAs and the monocot-prevalent reproductive phasiRNAs. Rather than giving a full review on anther development, here we discuss anther development with an emphasis on mobile elements like ROS/oxygen, secreted proteins and small RNAs (only briefly touching on phytohormones), how they might act and interact, and what the future of this research area might reveal.

A rapid method for quantification of persistent and mobile organic substances in water using supercritical fluid chromatography coupled to high-resolution mass spectrometry.

Persistent and mobile organic substances (PM substances) are a threat to the quality of our water resources. While screening studies revealed widespread occurrence of many PM substances, rapid trace analytical methods for their quantification in large sample sets are missing. We developed a quick and generic analytical method for highly mobile analytes in surface water, groundwater, and drinking water samples based on enrichment through azeotrope evaporation (4 mL water and 21 mL acetonitrile), supercritical fluid chromatography (SFC) coupled to high-resolution mass spectrometry (HRMS), and quantification using a compound-specific correction factor for apparent recovery. The method was validated using 17 PM substances. Sample preparation recoveries were between 60 and 110% for the vast majority of PM substances. Strong matrix effects (most commonly suppressive) were observed, necessitating a correction for apparent recoveries in quantification. Apparent recoveries were neither concentration dependent nor dependent on the water matrix (surface or drinking water). Method detection and quantification limits were in the single- to double-digit ng L-1 ranges, precision expressed as relative standard deviation of quadruplicate quantifications was on average < 10%, and trueness experiments showed quantitative results within ± 30% of the theoretical value in 77% of quantifications. Application of the method to surface water, groundwater, raw water, and finished drinking water revealed the presence of acesulfame and trifluoromethanesulfonic acid up to 70 and 19 µg L-1, respectively. Melamine, diphenylguanidine, p-dimethylbenzenesulfonic acid, and 4-hydroxy-1-(2-hydroxyethyl)-2,2,6,6-tetramethylpiperidine were found in high ng L-1 concentrations. Graphical abstract.

Corrigendum: Novel Meiotic miRNAs and Indications for a Role of PhasiRNAs in Meiosis.

[This corrects the article DOI: 10.3389/fpls.2016.00762.].

Isolating Male Meiocytes from Maize and Wheat for "-Omics" Analyses.

Genome-wide gene expression studies have become a routine approach due to the advances in sequencing technologies, their ease of use, and increasing affordability. Simultaneous investigation of small RNA expression adds further valuable information but is not adopted as widely yet. Both RNA-seq and small RNA-seq benefit from the use of specific cell types. Here, we describe a protocol for the isolation of male meiotic cells from maize or wheat plants, along with the application of downstream RNA sequencing, extendable to other -omics approaches.

The C4 Ppc promoters of many C4 grass species share a common regulatory mechanism for gene expression in the mesophyll cell.

C4 photosynthetic plants have evolved from C3 ancestors and are characterized by differential expression of several hundred genes. Strict compartmentalization of key C4 enzymes either to mesophyll (M) or bundle sheath cells is considered a crucial step towards the evolution of C4 photosynthesis. In this study, we demonstrate that the 5'-flanking sequences of the C4 type phosphoenolpyruvate carboxylase (Ppc) gene from three C4 grass species could drive M-cell-specific expression of a reporter gene in rice. In addition to that, we identified about 450 bp (upstream of their transcription start site) of the analyzed C4 Ppc promoters contain all the essential regulatory elements for driving M-cell-specific expression in rice leaves. Importantly, four motifs of conserved nucleotide sequences (CNSs) were also determined, which are essential for the activity of the promoter. A putative interaction between the CNSs and an unknown upstream element(s) is required for driving M-cell-specific expression. This work identifies the evolutionary conservation of C4 Ppc regulatory mechanisms of multiple closely related C4 grass species.

Targeted misexpression of NAC052, acting in H3K4 demethylation, alters leaf morphological and anatomical traits in Arabidopsis thaliana.

In an effort to identify genetic regulators for the cell ontogeny around the veins in Arabidopsis thaliana leaves, an activation-tagged mutant line with altered leaf morphology and altered bundle sheath anatomy was characterized. This mutant had a small rosette area with wrinkled leaves and chlorotic leaf edges, as well as enhanced chloroplast numbers in the (pre-)bundle sheath tissue. It had a bundle-specific promoter from the gene GLYCINE DECARBOXYLASE SUBUNIT-T from the C4 species Flaveria trinervia (GLDTFt promoter) inserted in the coding region of the transcriptional repressor NAC052, functioning in H3K4 demethylation, in front of an alternative start codon in-frame with the natural start codon. Reconstruction of the mutation event of our activation-tagged line by creating a line expressing an N-terminally truncated sequence of NAC052 under control of the GLDTFt promoter confirmed the involvement of NAC052 in leaf development. Our study not only reveals leaf anatomic and transcriptomic effects of an N-terminally truncated NAC052 under control of the GLDTFt promoter, but also identifies NAC052 as a novel genetic regulator of leaf development.

Correction to: MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat.

Following publication of the original article [1], a reader spotted an incorrect citation of the reference 14 [2] in the 'Background'. The male meiocyte isolation work described in this article [2] was carried out in rice and not in Brassica as originally stated in the 'Background' [1]. Thus, the following amendment to the Background section should be noted.

Occurrence of emerging persistent and mobile organic contaminants in European water samples.

The release of persistent and mobile organic chemicals (PMOCs) into the aquatic environment puts the quality of water resources at risk. PMOCs are challenging to analyze in water samples, due to their high polarity. The aim of this study was to develop novel analytical methods for PMOCs and to investigate their occurrence in surface and groundwater samples. The target compounds were culled from a prioritized list of industrial chemicals that were modeled to be persistent, mobile, and emitted into the environment. Analytical screening methods based on mixed-mode liquid chromatography (LC), hydrophilic interaction LC, reversed phase LC, or supercritical fluid chromatography in combination with mass spectrometric detection were successfully developed for 57 target PMOCs and applied to 14 water samples from three European countries. A total of 43 PMOCs were detected in at least one sample, among them 23 PMOCs that have not been reported before to occur in environmental waters. The most prevalent of these novel PMOCs were methyl sulfate, 2-acrylamino-2-methylpropane sulfonate, benzyltrimethylammonium, benzyldimethylamine, trifluoromethanesulfonic acid, 6-methyl-1,3,5-triazine-diamine, and 1,3-di-o-tolylguanidine occurring in ≥50% of the samples at estimated concentrations in the low ng L-1 up to µg L-1 range. The approach of focused prioritization combined with sensitive target chemical analysis proved to be highly efficient in revealing a large suite of novel as well as scarcely investigated PMOCs in surface and groundwater.

MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat.

BACKGROUND: Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks. RESULTS: We describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed. CONCLUSION: The MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species.

A Critical Assessment of 60 Years of Maize Intragenic Recombination.

Until the mid-1950s, it was believed that genetic crossovers did not occur within genes. Crossovers occurred between genes, the "beads on a string" model. Then in 1956, Seymour Benzer published his classic paper describing crossing over within a gene, intragenic recombination. This result from a bacteriophage gene prompted Oliver Nelson to study intragenic recombination in the maize Waxy locus. His studies along with subsequent work by others working with maize and other organisms described the outcomes of intragenic recombination and provided some of the earliest evidence that genes, not intergenic regions, were recombination hotspots. High-throughput genotyping approaches have since replaced single gene intragenic studies for characterizing the outcomes of recombination. These large-scale studies confirm that genes, or more generally genic regions, are the most active recombinogenic regions, and suggested a pattern of crossovers similar to the budding yeast Saccharomyces cerevisiae. In S. cerevisiae recombination is initiated by double-strand breaks (DSBs) near transcription start sites (TSSs) of genes producing a polarity gradient where crossovers preferentially resolve at the 5' end of genes. Intragenic studies in maize yielded less evidence for either polarity or for DSBs near TSSs initiating recombination and in certain respects resembled Schizosaccharomyces pombe or mouse. These different perspectives highlight the need to draw upon the strengths of different approaches and caution against relying on a single model system or approach for understanding recombination.

Using REACH registration data to rank the environmental emission potential of persistent and mobile organic chemicals.

Organic chemicals that are persistent and mobile in the aquatic environment exhibit a hazard to contaminate drinking water resources. In this study an emission score model was developed to rank the potential of substances registered under the REACH legislation to be emitted into the environment. It was applied to a list of 2167 REACH registered substances that were previously identified to be persistent and mobile organic chemicals (PMOCs) in groundwater or to be hydrolyzed to form transformation products fulfilling the PMOC criteria. The emission score model is based on the tonnage placed on the European market and on seven emission-related use characteristics (high release to environment, wide dispersive use, intermediate use, closed system use, professional use, consumer use, and substance in article), reported in the companies' registrations under REACH. Applying the model resulted in a list of 1110 substances (936 PMOCs and 174 precursors to PMOCs) that were estimated to be released into the environment, while 1054 substances had indicators of negligible environmental emissions and 3 substances could not be evaluated due to severe data gaps. The 936 PMOCs and the 174 precursors were ranked in two lists with regard to their emission potential. The model was shown to be fit for purpose in terms of suggesting and prioritizing substances for scientific investigations with a focus on environmental water quality. Though targeted for PMOCs, the presented scoring system is illustrative of how REACH registration data can be used to assess the emission potential of various substances.

High-resolution crossover mapping reveals similarities and differences of male and female recombination in maize.

Meiotic crossovers (COs) are not uniformly distributed across the genome. Factors affecting this phenomenon are not well understood. Although many species exhibit large differences in CO numbers between sexes, sex-specific aspects of CO landscape are particularly poorly elucidated. Here, we conduct high-resolution CO mapping in maize. Our results show that CO numbers as well as their overall distribution are similar in male and female meioses. There are, nevertheless, dissimilarities at local scale. Male and female COs differ in their locations relative to transcription start sites in gene promoters and chromatin marks, including nucleosome occupancy and tri-methylation of lysine 4 of histone H3 (H3K4me3). Our data suggest that sex-specific factors not only affect male-female CO number disparities but also cause fine differences in CO positions. Differences between male and female CO landscapes indicate that recombination has distinct implications for population structure and gene evolution in male and in female meioses.

Correction to: The transcriptome landscape of early maize meiosis.

CORRECTION: Following publication of the original article [1], the authors reported that the number of genes overlaying the bar graph in Fig. 3A were incorrectly counted and inserted (i.e. including a title tile, and in inverse order). The corrected numbers are below and match with the listed genes supplied in Additional File: Table S2.

Not just gene expression: 3D implications of chromatin modifications during sexual plant reproduction.

DNA methylation and histone modifications are epigenetic changes on a DNA molecule that alter the three-dimensional (3D) structure locally as well as globally, impacting chromatin looping and packaging on a larger scale. Epigenetic marks thus inform higher-order chromosome organization and placement in the nucleus. Conventional epigenetic marks are joined by chromatin modifiers like cohesins, condensins and membrane-anchoring complexes to support particularly 3D chromosome organization. The most popular consequences of epigenetic modifications are gene expression changes, but chromatin modifications have implications beyond this, particularly in actively dividing cells and during sexual reproduction. In this opinion paper, we will focus on epigenetic mechanisms and chromatin modifications during meiosis as part of plant sexual reproduction where 3D management of chromosomes and re-organization of chromatin are defining features and prime tasks in reproductive cells, not limited to modulating gene expression. Meiotic chromosome organization, pairing and synapsis of homologous chromosomes as well as distribution of meiotic double-strand breaks and resulting crossovers are presumably highly influenced by epigenetic mechanisms. Special mobile small RNAs have been described in anthers, where these so-called phasiRNAs seem to direct DNA methylation in meiotic cells. Intriguingly, many of the mentioned developmental processes make use of epigenetic changes and small RNAs in a manner other than gene expression changes. Widening our approaches and opening our mind to thinking three-dimensionally regarding epigenetics in plant development holds high promise for new discoveries and could give us a boost for further knowledge.