Cas9-targeted Nanopore sequencing rapidly elucidates the transposition preferences and DNA methylation profiles of mobile elements in plants.

Transposable element insertions (TEIs) are an important source of genomic innovation by contributing to plant adaptation, speciation, and the production of new varieties. The often large, complex plant genomes make identifying TEIs from short reads difficult and expensive. Moreover, rare somatic insertions that reflect mobilome dynamics are difficult to track using short reads. To address these challenges, we combined Cas9-targeted Nanopore sequencing (CANS) with the novel pipeline NanoCasTE to trace both genetically inherited and somatic TEIs in plants. We performed CANS of the EVADÉ (EVD) retrotransposon in wild-type Arabidopsis thaliana and rapidly obtained up to 40× sequence coverage. Analysis of hemizygous T-DNA insertion sites and genetically inherited insertions of the EVD transposon in the ddm1 (decrease in DNA methylation 1) genome uncovered the crucial role of DNA methylation in shaping EVD insertion preference. We also investigated somatic transposition events of the ONSEN transposon family, finding that genes that are downregulated during heat stress are preferentially targeted by ONSENs. Finally, we detected hypomethylation of novel somatic insertions for two ONSENs. CANS and NanoCasTE are effective tools for detecting TEIs and exploring mobilome organization in plants in response to stress and in different genetic backgrounds, as well as screening T-DNA insertion mutants and transgenic plants.

Improving focusability of post-compressed PW laser pulses using a deformable mirror.

The use of the post-compression technique ensures gain in laser pulse peak power but at the same time degrades beam focusability due to the nonlinear wavefront distortions caused by a spatially nonuniform beam profile. In this paper a substantial focusability improvement of a post-compressed laser pulse by means of adaptive optics was demonstrated experimentally. The Strehl ratio increase from 0.16 to 0.43 was measured. Simulations showed that the peak intensity in this case reaches 0.52 of the theoretical limit.

Genomic and Transcriptomic Survey Provides New Insight into the Organization and Transposition Activity of Highly Expressed LTR Retrotransposons of Sunflower (Helianthus annuus L.).

LTR retrotransposons (RTEs) play a crucial role in plant genome evolution and adaptation. Although RTEs are generally silenced in somatic plant tissues under non-stressed conditions, some expressed RTEs (exRTEs) escape genome defense mechanisms. As our understanding of exRTE organization in plants is rudimentary, we systematically surveyed the genomic and transcriptomic organization and mobilome (transposition) activity of sunflower (Helianthus annuus L.) exRTEs. We identified 44 transcribed RTEs in the sunflower genome and demonstrated their distinct genomic features: more recent insertion time, longer open reading frame (ORF) length, and smaller distance to neighboring genes. We showed that GAG-encoding ORFs are present at significantly higher frequencies in exRTEs, compared with non-expressed RTEs. Most exRTEs exhibit variation in copy number among sunflower cultivars and one exRTE Gagarin produces extrachromosomal circular DNA in seedling, demonstrating recent and ongoing transposition activity. Nanopore direct RNA sequencing of full-length RTE RNA revealed complex patterns of alternative splicing in RTE RNAs, resulting in isoforms that carry ORFs for distinct RTE proteins. Together, our study demonstrates that tens of expressed sunflower RTEs with specific genomic organization shape the hidden layer of the transcriptome, pointing to the evolution of specific strategies that circumvent existing genome defense mechanisms.

The wake- and sleep-modulating neurons of the lateral hypothalamic area demonstrate a differential pattern of degeneration in Alzheimers disease.

BACKGROUND: Sleep-wake dysfunction is an early and common event in Alzheimer's disease (AD). The lateral hypothalamic area (LHA) regulates the sleep and wake cycle through wake-promoting orexinergic neurons (OrxN) and sleep-promoting melanin-concentrating hormone or MCHergic neurons (MCHN). These neurons share close anatomical proximity with functional reciprocity. This study investigated LHA OrxN and MCHN loss patterns in AD individuals. Understanding the degeneration pattern of these neurons will be instrumental in designing potential therapeutics to slow down the disease progression and remediate the sleep-wake dysfunction in AD. METHODS: Postmortem human brain tissue from donors with AD (across progressive stages) and controls were examined using unbiased stereology. Formalin-fixed, celloidin-embedded hypothalamic sections were stained with Orx-A/MCH, p-tau (CP13), and counterstained with gallocyanin. Orx or MCH-positive neurons with or without CP13 inclusions and gallocyanin-stained neurons were considered for stereology counting. Additionally, we extracted RNA from the LHA using conventional techniques. We used customized Neuropathology and Glia nCounter (Nanostring) panels to study gene expression. Wald statistical test was used to compare the groups, and the genes were considered differentially expressed when the p-value was <.05. RESULTS: We observed a progressive decline in OrxN alongside a relative preservation of MCHN. OrxN decreased by 58% (p=0.03) by Braak stages (BB) 1-2 and further declined to 81% (p=0.03) by BB 5-6. Conversely, MCHN demonstrated a non-statistical significant decline (27%, p=0.1088) by BB 6. We observed a progressive increase in differentially expressed genes (DEGs), starting with glial profile changes in BB2. While OrxN loss was observed, Orx-related genes showed upregulation in BB 3-4 compared to BB 0-1. GO and KEGG terms related to neuroinflammatory pathways were mainly enriched. CONCLUSIONS: To date, OrxN loss in the LHA represents the first neuronal population to die preceding the loss of LC neurons. Conversely, MCHN shows resilience to AD p-tau accumulation across Braak stages. The initial loss of OrxN correlates with specific neuroinflammation, glial profile changes, and an overexpression of HCRT, possibly due to hyperexcitation following compensation mechanisms. Interventions preventing OrxN loss and inhibiting p-tau accumulation in the LHA could prevent neuronal loss in AD and, perhaps, the progression of the disease.

Vortical freak waves in water under external pressure action.

A vortical model for freak wave formation in water is presented. The wind action is simulated by nonuniform pressure on the free surface. The motion of the fluid is described by an exact solution of 2D hydrodynamic equations for ideal inviscid fluid in Lagrangian variables. Fluid particles rotate in circles of different radius. The model describes the appearance of a freak wave in the field of the Gerstner wave. The physical parameters of the wave and feasibility of the proposed scenario are discussed.

C. elegans patched-3 is an essential gene implicated in osmoregulation and requiring an intact permease transporter domain.

The nematode Caenorhabditis elegans has retained a rudimentary Hedgehog (Hh) signalling pathway; Hh and Smoothened (Smo) homologs are absent, but two highly related Patched gene homologs, ptc-1 and ptc-3, and 24 ptc-related (ptr) genes are present. We previously showed that ptc-1 is essential for germ line cytokinesis. Here, we report that ptc-3 is also an essential gene; the absence of ptc-3 results in a late embryonic lethality due to an apparent defect in osmoregulation. Rescue of a ptc-3 mutant with a ptc-3::gfp translational reporter reveals that ptc-3 is dynamically expressed in multiple tissues across development. Consistent with this pattern of expression, ptc-3(RNAi) reveals an additional postembryonic requirement for ptc-3 activity. Tissue-specific promoter studies indicate that hypodermal expression of ptc-3 is required for normal development. Missense changes in key residues of the sterol sensing domain (SSD) and the permease transporter domain GxxxD/E motif reveal that the transporter domain is essential for PTC-3 activity, whereas an intact SSD is dispensable. Taken together, our studies indicate that PTC proteins have retained essential roles in C. elegans that are independent of Smoothened (Smo). These observations reveal novel, and perhaps ancestral, roles for PTC proteins.

Hydrodynamic attractors in heavy ion collisions: a review.

A review of the recent progress of relativistic hydrodynamic attractors is presented, with a focus on applications in heavy ion collisions and the quark gluon plasma. Pedagogical introductions to the effective descriptions relevant for attractors in high energy physics, namely hydrodynamics, holography and kinetic theory, are followed by highlights of some recent advances.

Successful Silencing of the Mycotoxin Synthesis Gene TRI5 in Fusarium culmorum and Observation of Reduced Virulence in VIGS and SIGS Experiments.

Crops constantly experience various biotic stresses during their life cycle, and Fusarium spp. remain one of the most serious groups of pathogens affecting plants. The ability to manipulate the expression of certain microorganism genes via RNAi creates the opportunity for new-generation dsRNA-based preparations to control a large number of diseases. In this study, we applied virus-induced gene silencing (VIGS), and spray-induced gene silencing (SIGS) to silence the trichothecene-producing gene TRI5 in F. culmorum as a means to reduce its aggressiveness on spring wheat. Treatment of the fungus with dsTRI5RNA in vitro reduced deoxynivalenol (DON) and 3-acetyldeoxynivalenol (3-A-DON) accumulations by 53-85% and 61-87%, respectively, and reduced TRI5 expression by 84-97%. VIGS decreased the proportion of infected wheat spikelets by 73%, but upregulation was observed for TRI5. SIGS on wheat leaves and ears using certain dsTRI5RNA amounts negatively impacted F. culmorum growth. However, when performing in vivo analyses of TRI5 mRNA levels, the upregulation of the gene was determined in the variants where fungal colonization was restricted, suggesting a compensatory reaction of the pathogen to RNAi.

Epigenetic Stress and Long-Read cDNA Sequencing of Sunflower (Helianthus annuus L.) Revealed the Origin of the Plant Retrotranscriptome.

Transposable elements (TEs) contribute not only to genome diversity but also to transcriptome diversity in plants. To unravel the sources of LTR retrotransposon (RTE) transcripts in sunflower, we exploited a recently developed transposon activation method ('TEgenesis') along with long-read cDNA Nanopore sequencing. This approach allows for the identification of 56 RTE transcripts from different genomic loci including full-length and non-autonomous RTEs. Using the mobilome analysis, we provided a new set of expressed and transpositional active sunflower RTEs for future studies. Among them, a Ty3/Gypsy RTE called SUNTY3 exhibited ongoing transposition activity, as detected by eccDNA analysis. We showed that the sunflower genome contains a diverse set of non-autonomous RTEs encoding a single RTE protein, including the previously described TR-GAG (terminal repeat with the GAG domain) as well as new categories, TR-RT-RH, TR-RH, and TR-INT-RT. Our results demonstrate that 40% of the loci for RTE-related transcripts (nonLTR-RTEs) lack their LTR sequences and resemble conventional eucaryotic genes encoding RTE-related proteins with unknown functions. It was evident based on phylogenetic analysis that three nonLTR-RTEs encode GAG (HadGAG1-3) fused to a host protein. These HadGAG proteins have homologs found in other plant species, potentially indicating GAG domestication. Ultimately, we found that the sunflower retrotranscriptome originated from the transcription of active RTEs, non-autonomous RTEs, and gene-like RTE transcripts, including those encoding domesticated proteins.

Searching for a Needle in a Haystack: Cas9-Targeted Nanopore Sequencing and DNA Methylation Profiling of Full-Length Glutenin Genes in a Big Cereal Genome.

Sequencing and epigenetic profiling of target genes in plants are important tasks with various applications ranging from marker design for plant breeding to the study of gene expression regulation. This is particularly interesting for plants with big genome size for which whole-genome sequencing can be time-consuming and costly. In this study, we asked whether recently proposed Cas9-targeted nanopore sequencing (nCATS) is efficient for target gene sequencing for plant species with big genome size. We applied nCATS to sequence the full-length glutenin genes (Glu-1Ax, Glu-1Bx and Glu-1By) and their promoters in hexaploid triticale (X Triticosecale, AABBRR, genome size is 24 Gb). We showed that while the target gene enrichment per se was quite high for the three glutenin genes (up to 645×), the sequencing depth that was achieved from two MinION flowcells was relatively low (5-17×). However, this sequencing depth was sufficient for various tasks including detection of InDels and single-nucleotide variations (SNPs), read phasing and methylation profiling. Using nCATS, we uncovered SNP and InDel variation of full-length glutenin genes providing useful information for marker design and deciphering of variation of individual Glu-1By alleles. Moreover, we demonstrated that glutenin genes possess a 'gene-body' methylation epigenetic profile with hypermethylated CDS part and hypomethylated promoter region. The obtained information raised an interesting question on the role of gene-body methylation in glutenin gene expression regulation. Taken together, our work disclosures the potential of the nCATS approach for sequencing of target genes in plants with big genome size.

Transposons Hidden in Arabidopsis thaliana Genome Assembly Gaps and Mobilization of Non-Autonomous LTR Retrotransposons Unravelled by Nanotei Pipeline.

Long-read data is a great tool to discover new active transposable elements (TEs). However, no ready-to-use tools were available to gather this information from low coverage ONT datasets. Here, we developed a novel pipeline, nanotei, that allows detection of TE-contained structural variants, including individual TE transpositions. We exploited this pipeline to identify TE insertion in the Arabidopsis thaliana genome. Using nanotei, we identified tens of TE copies, including ones for the well-characterized ONSEN retrotransposon family that were hidden in genome assembly gaps. The results demonstrate that some TEs are inaccessible for analysis with the current A. thaliana (TAIR10.1) genome assembly. We further explored the mobilome of the ddm1 mutant with elevated TE activity. Nanotei captured all TEs previously known to be active in ddm1 and also identified transposition of non-autonomous TEs. Of them, one non-autonomous TE derived from (AT5TE33540) belongs to TR-GAG retrotransposons with a single open reading frame (ORF) encoding the GAG protein. These results provide the first direct evidence that TR-GAGs and other non-autonomous LTR retrotransposons can transpose in the plant genome, albeit in the absence of most of the encoded proteins. In summary, nanotei is a useful tool to detect active TEs and their insertions in plant genomes using low-coverage data from Nanopore genome sequencing.

Nanopore RNA Sequencing Revealed Long Non-Coding and LTR Retrotransposon-Related RNAs Expressed at Early Stages of Triticale SEED Development.

The intergenic space of plant genomes encodes many functionally important yet unexplored RNAs. The genomic loci encoding these RNAs are often considered "junk", DNA as they are frequently associated with repeat-rich regions of the genome. The latter makes the annotations of these loci and the assembly of the corresponding transcripts using short RNAseq reads particularly challenging. Here, using long-read Nanopore direct RNA sequencing, we aimed to identify these "junk" RNA molecules, including long non-coding RNAs (lncRNAs) and transposon-derived transcripts expressed during early stages (10 days post anthesis) of seed development of triticale (AABBRR, 2n = 6x = 42), an interspecific hybrid between wheat and rye. Altogether, we found 796 lncRNAs and 20 LTR retrotransposon-related transcripts (RTE-RNAs) expressed at this stage, with most of them being previously unannotated and located in the intergenic as well as intronic regions. Sequence analysis of the lncRNAs provide evidence for the frequent exonization of Class I (retrotransposons) and class II (DNA transposons) transposon sequences and suggest direct influence of "junk" DNA on the structure and origin of lncRNAs. We show that the expression patterns of lncRNAs and RTE-related transcripts have high stage specificity. In turn, almost half of the lncRNAs located in Genomes A and B have the highest expression levels at 10-30 days post anthesis in wheat. Detailed analysis of the protein-coding potential of the RTE-RNAs showed that 75% of them carry open reading frames (ORFs) for a diverse set of GAG proteins, the main component of virus-like particles of LTR retrotransposons. We further experimentally demonstrated that some RTE-RNAs originate from autonomous LTR retrotransposons with ongoing transposition activity during early stages of triticale seed development. Overall, our results provide a framework for further exploration of the newly discovered lncRNAs and RTE-RNAs in functional and genome-wide association studies in triticale and wheat. Our study also demonstrates that Nanopore direct RNA sequencing is an indispensable tool for the elucidation of lncRNA and retrotransposon transcripts.

Functional Allium fistulosum Centromeres Comprise Arrays of a Long Satellite Repeat, Insertions of Retrotransposons and Chloroplast DNA.

The centromere is a unique part of the chromosome combining a conserved function with an extreme variability in its DNA sequence. Most of our knowledge about the functional centromere organization is obtained from species with small and medium genome/chromosome sizes while the progress in plants with big genomes and large chromosomes is lagging behind. Here, we studied the genomic organization of the functional centromere in Allium fistulosum and A. cepa, both species with a large genome (13 Gb and 16 Gb/1C, 2n = 2x = 16) and large-sized chromosomes. Using low-depth DNA sequencing for these two species and previously obtained CENH3 immunoprecipitation data we identified two long (1.2 Kb) and high-copy repeats, AfCen1K and AcCen1K. FISH experiments showed that AfCen1K is located in all centromeres of A. fistulosum chromosomes while no AcCen1K FISH signals were identified on A. cepa chromosomes. Our molecular cytogenetic and bioinformatics survey demonstrated that these repeats are partially similar but differ in chromosomal location, sequence structure and genomic organization. In addition, we could conclude that the repeats are transcribed and their RNAs are not polyadenylated. We also observed that these repeats are associated with insertions of retrotransposons and plastidic DNA and the landscape of A. cepa and A. fistulosum centromeric regions possess insertions of plastidic DNA. Finally, we carried out detailed comparative satellitome analysis of A. cepa and A. fistulosum genomes and identified a new chromosome- and A. cepa-specific tandem repeat, TR2CL137, located in the centromeric region. Our results shed light on the Allium centromere organization and provide unique data for future application in Allium genome annotation.

Potential effect of bio-surfactants on sea spray generation in tropical cyclone conditions.

Despite significant improvement in computational and observational capabilities, predicting intensity and intensification of major tropical cyclones remains a challenge. In 2017 Hurricane Maria intensified to a Category 5 storm within 24 h, devastating Puerto Rico. In 2019 Hurricane Dorian, predicted to remain tropical storm, unexpectedly intensified into a Category 5 storm and destroyed the Bahamas. The official forecast and computer models were unable to predict rapid intensification of these storms. One possible reason for this is that key physics, including microscale processes at the air-sea interface, are poorly understood and parameterized in existing forecast models. Here we show that surfactants significantly affect the generation of sea spray, which provides some of the fuel for tropical cyclones and their intensification, but also provides some of the drag that limits intensity and intensification. Using a numerical model verified with a laboratory experiment, which predicts spray radii distribution starting from a 100 µm radius, we show that surfactants increase spray generation by 20-34%. We anticipate that bio-surfactants affect heat, energy, and momentum exchange through altered size distribution and concentration of sea spray, with consequences for tropical cyclone intensification or decline, particularly in areas of algal blooms and near coral reefs, as well as in areas affected by oil spills and dispersants.

Effect of Dwarfing Gene Ddw1 on Height and Agronomic Traits in Spring Triticale in Greenhouse and Field Experiments in a Non-Black Earth Region of Russia.

Triticale is a relatively new crop which still possesses serious drawbacks that can be significantly improved by breeding. The dwarfing genes proved to be very useful in the development of new lodging resistant and productive cultivars of winter triticale. The aim of our research was to assess the effect of the Ddw1 dwarfing gene from rye on the agronomic valuable traits in spring triticale. The Ddw1 effect was studied in the greenhouse experiment in segregating the F2:3 population and in the field of F3:4 and F4:5 families derived from crossing winter triticale 'Hongor' (Ddw1Ddw1) and spring triticale 'Dublet' (ddw1ddw1). As a result, in all three generations, a strong decrease in plant height was demonstrated that was accompanied by a decrease in grain weight per spike and 1000-grain weight. In field experiments, a decrease in spike length and increase in spike density and delay in flowering and heading were observed. As a result of decrease in culm vegetative weight due to Ddw1, the harvest index measured in F4:5 increased. The spike fertility and number of grains were not affected by Ddw1. The comparison of Ddw1 in rye, winter, and spring triticale, and the possible role of Ddw1 in improving spring triticale are discussed.

Experimental study of thermal lens features in laser ceramics.

Thermal lens measurements were made by means of a high-accuracy phase shift interferometer that combines a lambda/1000 sensitivity and 10 microm transverse resolution. The effect of random small-scale modulation in thermally induced phase distortion predicted earlier was proved experimentally. The statistical parameters of modulation were measured depending on heating power for two different ceramic samples. The experimental data agree well with results of numerical simulation.

Is the State of the Air-Sea Interface a Factor in Rapid Intensification and Rapid Decline of Tropical Cyclones?

Tropical storm intensity prediction remains a challenge in tropical meteorology. Some tropical storms undergo dramatic rapid intensification and rapid decline. Hurricane researchers have considered particular ambient environmental conditions including the ocean thermal and salinity structure and internal vortex dynamics (e.g., eyewall replacement cycle, hot towers) as factors creating favorable conditions for rapid intensification. At this point, however, it is not exactly known to what extent the state of the sea surface controls tropical cyclone dynamics. Theoretical considerations, laboratory experiments, and numerical simulations suggest that the air-sea interface under tropical cyclones is subject to the Kelvin-Helmholtz type instability. Ejection of large quantities of spray particles due to this instability can produce a two-phase environment, which can attenuate gravity-capillary waves and alter the air-sea coupling. The unified parameterization of waveform and two-phase drag based on the physics of the air-sea interface shows the increase of the aerodynamic drag coefficient Cd with wind speed up to hurricane force ( U10≈35 m s-1). Remarkably, there is a local Cd minimum-"an aerodynamic drag well"-at around U10≈60 m s-1. The negative slope of the Cd dependence on wind-speed between approximately 35 and 60 m s-1 favors rapid storm intensification. In contrast, the positive slope of Cd wind-speed dependence above 60 m s-1 is favorable for a rapid storm decline of the most powerful storms. In fact, the storms that intensify to Category 5 usually rapidly weaken afterward.

DRAWID: user-friendly java software for chromosome measurements and idiogram drawing.

An idiogram construction following chromosome measurements is a versatile tool for cytological, cytogenetic and phylogenetic studies. The information on chromosome length, centromere index and position of cytogenetic landmarks along with modern techniques (e.g. genomic and fluorescence in situ hybridization, banding, chromosome painting) can help to shed light on genome constitution, chromosome rearrangements and evolution. While idiogram construction is a routine task there are only few freely available programs that can perform chromosome measurements and no software for simultaneous measuring of chromosome parameters, chromosomal landmark and FISH signal positions and idiogram construction. To fill this gap, we developed DRAWID (DRAWing IDiogram), java-based cross-platforming program for chromosome analysis and idiogram construction. DRAWID has number of advantages including a user-friendly interactive interface, possibility for simultaneous chromosome and FISH/GISH/banding signal measurement and idiogram drawing as well as number of useful functions facilitating the procedure of chromosome analysis. The output of the program is Microsoft XL table and publish-ready idiogram picture. DRAWID and the manual for its use are freely available on the website at: http://www.drawid.xyz.

Laboratory unraveling of matter accretion in young stars.

Accretion dynamics in the formation of young stars is still a matter of debate because of limitations in observations and modeling. Through scaled laboratory experiments of collimated plasma accretion onto a solid in the presence of a magnetic field, we open a first window on this phenomenon by tracking, with spatial and temporal resolution, the dynamics of the system and simultaneously measuring multiband emissions. We observe in these experiments that matter, upon impact, is ejected laterally from the solid surface and then refocused by the magnetic field toward the incoming stream. This ejected matter forms a plasma shell that envelops the shocked core, reducing escaped x-ray emission. This finding demonstrates one possible structure reconciling current discrepancies between mass accretion rates derived from x-ray and optical observations, respectively.