Pixelated High-Q Metasurfaces for in Situ Biospectroscopy and Artificial Intelligence-Enabled Classification of Lipid Membrane Photoswitching Dynamics.

Nanophotonic devices excel at confining light into intense hot spots of electromagnetic near fields, creating exceptional opportunities for light-matter coupling and surface-enhanced sensing. Recently, all-dielectric metasurfaces with ultrasharp resonances enabled by photonic bound states in the continuum (BICs) have unlocked additional functionalities for surface-enhanced biospectroscopy by precisely targeting and reading out the molecular absorption signatures of diverse molecular systems. However, BIC-driven molecular spectroscopy has so far focused on end point measurements in dry conditions, neglecting the crucial interaction dynamics of biological systems. Here, we combine the advantages of pixelated all-dielectric metasurfaces with deep learning-enabled feature extraction and prediction to realize an integrated optofluidic platform for time-resolved in situ biospectroscopy. Our approach harnesses high-Q metasurfaces specifically designed for operation in a lossy aqueous environment together with advanced spectral sampling techniques to temporally resolve the dynamic behavior of photoswitchable lipid membranes. Enabled by a software convolutional neural network, we further demonstrate the real-time classification of the characteristic cis and trans membrane conformations with 98% accuracy. Our synergistic sensing platform incorporating metasurfaces, optofluidics, and deep learning reveals exciting possibilities for studying multimolecular biological systems, ranging from the behavior of transmembrane proteins to the dynamic processes associated with cellular communication.

Origin and diversity of Capsella bursa-pastoris from the genomic point of view.

BACKGROUND: Capsella bursa-pastoris, a cosmopolitan weed of hybrid origin, is an emerging model object for the study of early consequences of polyploidy, being a fast growing annual and a close relative of Arabidopsis thaliana. The development of this model is hampered by the absence of a reference genome sequence. RESULTS: We present here a subgenome-resolved chromosome-scale assembly and a genetic map of the genome of Capsella bursa-pastoris. It shows that the subgenomes are mostly colinear, with no massive deletions, insertions, or rearrangements in any of them. A subgenome-aware annotation reveals the lack of genome dominance-both subgenomes carry similar number of genes. While most chromosomes can be unambiguously recognized as derived from either paternal or maternal parent, we also found homeologous exchange between two chromosomes. It led to an emergence of two hybrid chromosomes; this event is shared between distant populations of C. bursa-pastoris. The whole-genome analysis of 119 samples belonging to C. bursa-pastoris and its parental species C. grandiflora/rubella and C. orientalis reveals introgression from C. orientalis but not from C. grandiflora/rubella. CONCLUSIONS: C. bursa-pastoris does not show genome dominance. In the earliest stages of evolution of this species, a homeologous exchange occurred; its presence in all present-day populations of C. bursa-pastoris indicates on a single origin of this species. The evidence coming from whole-genome analysis challenges the current view that C. grandiflora/rubella was a direct progenitor of C. bursa-pastoris; we hypothesize that it was an extinct (or undiscovered) species sister to C. grandiflora/rubella.

Real-time simultaneous refractive index and thickness mapping of sub-cellular biology at the diffraction limit.

Mapping the cellular refractive index (RI) is a central task for research involving the composition of microorganisms and the development of models providing automated medical screenings with accuracy beyond 95%. These models require significantly enhancing the state-of-the-art RI mapping capabilities to provide large amounts of accurate RI data at high throughput. Here, we present a machine-learning-based technique that obtains a biological specimen's real-time RI and thickness maps from a single image acquired with a conventional color camera. This technology leverages a suitably engineered nanostructured membrane that stretches a biological analyte over its surface and absorbs transmitted light, generating complex reflection spectra from each sample point. The technique does not need pre-existing sample knowledge. It achieves 10-4 RI sensitivity and sub-nanometer thickness resolution on diffraction-limited spatial areas. We illustrate practical application by performing sub-cellular segmentation of HCT-116 colorectal cancer cells, obtaining complete three-dimensional reconstruction of the cellular regions with a characteristic length of 30 µm. These results can facilitate the development of real-time label-free technologies for biomedical studies on microscopic multicellular dynamics.

The genome of the toxic invasive species Heracleum sosnowskyi carries an increased number of genes despite absence of recent whole-genome duplications.

Heracleum sosnowskyi, belonging to a group of giant hogweeds, is a plant with large effects on ecosystems and human health. It is an invasive species that contributes to the deterioration of grassland ecosystems. The ability of H. sosnowskyi to produce linear furanocoumarins (FCs), photosensitizing compounds, makes it very dangerous. At the same time, linear FCs are compounds with high pharmaceutical value used in skin disease therapies. Despite this high importance, it has not been the focus of genetic and genomic studies. Here, we report a chromosome-scale assembly of Sosnowsky's hogweed genome. Genomic analysis revealed an unusually high number of genes (55106) in the hogweed genome, in contrast to the 25-35 thousand found in most plants. However, we did not find any traces of recent whole-genome duplications not shared with its confamiliar, Daucus carota (carrot), which has approximately thirty thousand genes. The analysis of the genomic proximity of duplicated genes indicates on tandem duplications as a main reason for this increase. We performed a genome-wide search of the genes of the FC biosynthesis pathway and surveyed their expression in aboveground plant parts. Using a combination of expression data and phylogenetic analysis, we found candidate genes for psoralen synthase and experimentally showed the activity of one of them using a heterologous yeast expression system. These findings expand our knowledge on the evolution of gene space in plants and lay a foundation for further analysis of hogweed as an invasive plant and as a source of FCs.

Origin of CMS-PET1 cytotype in cultivated sunflower: A new insight.

The vast majority of commercial sunflower hybrids worldwide are produced using cytoplasmic male sterility (CMS) of the PET1 type, resulting from the interspecific hybridization of Helianthus petiolaris with Helianthus annuus. Due to the fact that CMS-PET1 was not previously detected in wild sunflower, it was believed that this cytotype could arise during interspecific hybridization and is specific solely for cultivated sunflower. In this study, the open reading frame, orfH522, associated with the CMS-PET1 phenotype, was revealed for the first time in the 3'-flanking region of the mitochondrial atpA gene in wild H. annuus. An analysis of whole genome data from 1089 accessions showed that the frequency of occurrence of CMS-orfH522 in wild H. annuus populations is 3.58%, while in wild H. petiolaris populations, it is 1.26%. In general, the analysis demonstrated that PET1-CMS is a natural cytotype of H. annuus, and the appearance of the CMS phenotype in cultivated sunflowers is associated with the loss of stabilizing nuclear genes of fertility restorers, which occurred during interspecific hybridization. These data can explain the patterns of differential cytoplasmic and nuclear introgression occurring in wild sunflower and are useful for further evolutionary studies.

Mitogenomic Research of Silverleaf Sunflower (Helianthus argophyllus) and Its Interspecific Hybrids.

Interspecific hybridization is widespread for sunflowers, both in wild populations and commercial breeding. One of the most common species that can efficiently cross with Helianthus annuus is the Silverleaf sunflower-Helianthus argophyllus. The current study carried out structural and functional organization analyses of mitochondrial DNA in H. argophyllus and the interspecific hybrid, H. annuus (VIR114A line) × H. argophyllus. The complete mitogenome of H. argophyllus counts 300,843 bp, has a similar organization to the mitogenome of cultivated sunflower, and holds SNPs typical for wild sunflowers. RNA editing analysis predicted 484 sites in H. argophyllus mitochondrial CDS. The mitochondrial genome of the H. annuus × H. argophyllus hybrid is identical to the maternal line (VIR114A). We expected that significant rearrangements in the mitochondrial DNA of the hybrid would occur, due to the frequent recombination. However, the hybrid mitogenome lacks rearrangements, presumably due to the preservation of nuclear-cytoplasmic interaction paths.

The Insights into Mitochondrial Genomes of Sunflowers.

The significant difference in the mtDNA size and structure with simultaneous slow evolving genes makes the mitochondrial genome paradoxical among all three DNA carriers in the plant cell. Such features make mitochondrial genome investigations of particular interest. The genus Helianthus is a diverse taxonomic group, including at least two economically valuable species-common sunflower (H. annuus) and Jerusalem artichoke (H. tuberosus). The successful investigation of the sunflower nuclear genome provided insights into some genomics aspects and significantly intensified sunflower genetic studies. However, the investigations of organelles' genetic information in Helianthus, especially devoted to mitochondrial genomics, are presented by limited studies. Using NGS sequencing, we assembled the complete mitochondrial genomes for H. occidentalis (281,175 bp) and H. tuberosus (281,287 bp) in the current investigation. Besides the master circle chromosome, in the case of H. tuberosus, the 1361 bp circular plasmid was identified. The mitochondrial gene content was found to be identical for both sunflower species, counting 32 protein-coding genes, 3 rRNA, 23 tRNA genes, and 18 ORFs. The comparative analysis between perennial sunflowers revealed common and polymorphic SSR and SNPs. Comparison of perennial sunflowers with H. annuus allowed us to establish similar rearrangements in mitogenomes, which have possibly been inherited from a common ancestor after the divergence of annual and perennial sunflower species. It is notable that H. occidentalis and H. tuberosus mitogenomes are much more similar to H. strumosus than H. grosseserratus.

Data on the polymorphic sites in the chloroplast genomes of seven perennial Helianthus species.

Data present the chloroplast genome sequences of seven wild perennial Helianthus species obtained by using the Illumina HiSeq and NextSeq platforms. Datasets not included in the primary publication [1] are a source for further evolutionary studies. In particular, the annotated chloroplast genomes and datasets of single nucleotide polymorphisms (SNP), simple sequence repeats (SSR), insertion and deletion polymorphisms (INDEL) for H. tuberosus, H. salicifolius, H. pauciflorus, H. microcephalis, H. hirsutus, H. strumosus, and H. grosseserratus are presented. The raw reads are available in Figshare (https://doi.org/10.6084/m9.figshare.12600155). The complete chloroplast genome sequences for the seven perennial Helianthus species are available on GenBank NCBI under the accessions MT302562.1 - MT302568.1; the remaining data are provided in this article.

Comparative analysis of chloroplast genomes of seven perennial Helianthus species.

Sequencing and a comparative analysis of the complete chloroplast genomes of seven perennial Helianthus species were carried out. The chloroplast genomes have a typical quadripartite structure, including large and small single regions and a pair of inverted repeats. Genome sizes were between 151,152 bp and 151,289 bp. The genome of H. grosseserratus was the smallest, while that of H. microcephalus was the largest. The size variation of the chloroplast genomes is substantially due to the change in the length of simple sequence repeats (SSRs) in non-coding regions. An analysis of these SSRs revealed 35 polymorphic loci (average PIC value > 0.5) that can be used to examine ecological and evolutionary processes in wild Helianthus species. Eight divergence hotspots, including five intergenic regions (petN-psbM, clpP intron, rps3-rpl16, ndhD-ccsA, and ndhF-rpl32) and three gene regions (rbcL, ycf1, and ndhF) were also identified in Helianthus chloroplast genomes. The evolutionary selection pressure analysis revealed a strong purifying selection. Only the rbcL gene experienced efficiency of positive selection at the annual/perennial transitions. The inverted repeat (IR)/single copy (SC) boundaries were identical in all of these (Helianthus) species. In general, the comparison of the genomes revealed low levels of sequence variability (Pi = 0.00051). This indicates that the chloroplast genomes of the studied perennial species of Helianthus, in addition to purifying selection, are closely related and have a recent divergence time.

The Investigation of Perennial Sunflower Species (Helianthus L.) Mitochondrial Genomes.

The genus Helianthus is a diverse taxonomic group with approximately 50 species. Most sunflower genomic investigations are devoted to economically valuable species, e.g., H. annuus, while other Helianthus species, especially perennial, are predominantly a blind spot. In the current study, we have assembled the complete mitogenomes of two perennial species: H. grosseserratus (273,543 bp) and H. strumosus (281,055 bp). We analyzed their sequences and gene profiles in comparison to the available complete mitogenomes of H. annuus. Except for sdh4 and trnA-UGC, both perennial sunflower species had the same gene content and almost identical protein-coding sequences when compared with each other and with annual sunflowers (H. annuus). Common mitochondrial open reading frames (ORFs) (orf117, orf139, and orf334) in sunflowers and unique ORFs for H. grosseserratus (orf633) and H. strumosus (orf126, orf184, orf207) were identified. The maintenance of plastid-derived coding sequences in the mitogenomes of both annual and perennial sunflowers and the low frequency of nonsynonymous mutations point at an extremely low variability of mitochondrial DNA (mtDNA) coding sequences in the Helianthus genus.

A point mutation in the photosystem I P700 chlorophyll a apoprotein A1 gene confers variegation in Helianthus annuus L.

KEY MESSAGE: Even a point mutation in the psaA gene mediates chlorophyll deficiency. The role of the plastid signal may perform the redox state of the compounds on the acceptor-side of PSI. Two extranuclear variegated mutants of sunflower, Var1 and Var33, were investigated. The yellow sectors of both mutants were characterized by an extremely low chlorophyll and carotenoid content, as well as poorly developed, unstacked thylakoid membranes. A full-genome sequencing of the cpDNA revealed mutations in the psaA gene in both Var1 and Var33. The cpDNA from the yellow sectors of Var1 differs from those in the wild type by only a single, non-synonymous substitution (Gly734Glu) in the psaA gene, which encodes a subunit of photosystem (PS) I. In the cpDNA from the yellow sectors of Var33, the single-nucleotide insertion in the psaA gene was revealed, leading to frameshift at the 580 amino acid position. Analysis of the photosynthetic electron transport demonstrated an inhibition of the PSI and PSII activities in the yellow tissues of the mutant plants. It has been suggested that mutations in the psaA gene of both Var1 and Var33 led to the disruption of PSI. Due to the non-functional PSI, photosynthetic electron transport is blocked, which, in turn, leads to photodamage of PSII. These data are confirmed by immunoblotting analysis, which showed a significant reduction in PsbA in the yellow leaf sectors, but not PsaA. The expression of chloroplast and nuclear genes encoding the PSI subunits (psaA, psaB, and PSAN), the PSII subunits (psbA, psbB, and PSBW), the antenna proteins (LHCA1, LHCB1, and LHCB4), the ribulose 1.5-bisphosphate carboxylase subunits (rbcL and RbcS), and enzymes of chlorophyll biosynthesis were down-regulated in the yellow leaf tissue. The extremely reduced transcriptional activity of the two protochlorophyllide oxidoreductase (POR) genes involved in chlorophyll biosynthesis is noteworthy. The disruption of NADPH synthesis, due to the non-functional PSI, probably led to a significant reduction in NADPH-protochlorophyllide oxidoreductase in the yellow sectors of Var1 and Var33. A dramatic decrease in chlorophyllide was shown in the yellow sectors. A reduction in NADPH-protochlorophyllide oxidoreductase, along with photodegradation, has been suggested as a result of chlorophyll deficiency.

Organization Features of the Mitochondrial Genome of Sunflower (Helianthus annuus L.) with ANN2-Type Male-Sterile Cytoplasm.

This study provides insights into the flexibility of the mitochondrial genome in sunflower (Helianthus annuus L.) as well as into the causes of ANN2-type cytoplasmic male sterility (CMS). De novo assembly of the mitochondrial genome of male-sterile HA89(ANN2) sunflower line was performed using high-throughput sequencing technologies. Analysis of CMS ANN2 mitochondrial DNA sequence revealed the following reorganization events: twelve rearrangements, seven insertions, and nine deletions. Comparisons of coding sequences from the male-sterile line with the male-fertile line identified a deletion of orf777 and seven new transcriptionally active open reading frames (ORFs): orf324, orf327, orf345, orf558, orf891, orf933, orf1197. Three of these ORFs represent chimeric genes involving atp6 (orf1197), cox2 (orf558), and nad6 (orf891). In addition, orf558, orf891, orf1197, as well as orf933, encode proteins containing membrane domain(s), making them the most likely candidate genes for CMS development in ANN2. Although the investigated CMS phenotype may be caused by simultaneous action of several candidate genes, we assume that orf1197 plays a major role in developing male sterility in ANN2. Comparative analysis of mitogenome organization in sunflower lines representing different CMS sources also allowed identification of reorganization hot spots in the mitochondrial genome of sunflower.

Data on the polymorphic sites in the chloroplast genomes of the sunflower alloplasmic CMS lines.

Data presents the chloroplast genome sequences of the five sunflower alloplasmic cytoplasmic male sterility (CMS) lines obtained with using the Illumina MiSeq, HiSeq and NextSeq platforms. The sunflower alloplasmic CMS lines has the same nuclear genome from line HA89, but they differ in cytoplasmic genomes, inherited from annual (PET1, PET2 - H. petiolaris, ANN2 - H. annuus) and perennial (MAX1 - H. maximilliani) species of the genus Helianthus L. The chloroplast genomes were annotated. Also presented is a dataset of variable sites such as single nucleotide polymorphism (SNP), simple sequence repeat (SSR), insertion and deletion (INDEL) in the chloroplast genome of the sequenced alloplasmic lines. The raw reads are available in FIGSHARE (https://doi.org/10.6084/m9.figshare.7520183). The complete chloroplast genome sequences for the sunflower alloplasmic lines are available in GenBank NCBI under the accessions MK341448.1-MK341452.1; the remaining data are provided with this article.

Cyclohexane, naphthalene, and diesel fuel increase oxidative stress, CYP153, sodA, and recA gene expression in Rhodococcus erythropolis.

In this study, we compared the expression of CYP153, sodA, sodC, and recA genes and ROS generation in hydrocarbon-degrading Rhodococcus erythropolis in the presence of cyclohexane, naphthalene, and diesel fuel. The expression of cytochrome P450, sodA (encoding Fe/Mn superoxide dismutase), recA, and superoxide anion radical generation rate increased after the addition of all studied hydrocarbons. The peak of CYP153, sodA, and recA gene expression was registered in the presence of naphthalene. The same substrate upregulated the Cu/Zn superoxide dismutase gene, sodC. Cyclohexane generated the highest level of superoxide anion radical production. Hydrogen peroxide accumulated in the medium enriched with diesel fuel. Taken together, hydrocarbon biotransformation leads to oxidative stress and upregulation of antioxidant enzymes and CYP153 genes, and increases DNA reparation levels in R. erythropolis cells.

Characterization of the mitochondrial genome of the MAX1 type of cytoplasmic male-sterile sunflower.

BACKGROUND: More than 70 cytoplasmic male sterility (CMS) types have been identified in Helianthus, but only for less than half of them, research of mitochondrial organization has been conducted. Moreover, complete mitochondrion sequences have only been published for two CMS sources - PET1 and PET2. It has been demonstrated that other sunflower CMS sources like MAX1, significantly differ from the PET1 and PET2 types. However, possible molecular causes for the CMS induction by MAX1 have not yet been proposed. In the present study, we have investigated structural changes in the mitochondrial genome of HA89 (MAX1) CMS sunflower line in comparison to the fertile mitochondrial genome. RESULTS: Eight significant major reorganization events have been determined in HA89 (MAX1) mtDNA: one 110 kb inverted region, four deletions of 439 bp, 978 bp, 3183 bp and 14,296 bp, respectively, and three insertions of 1999 bp, 5272 bp and 6583 bp. The rearrangements have led to functional changes in the mitochondrial genome of HA89 (MAX1) resulting in the complete elimination of orf777 and the appearance of new ORFs - orf306, orf480, orf645 and orf1287. Aligning the mtDNA of the CMS sources PET1 and PET2 with MAX1 we found some common reorganization features in their mitochondrial genome sequences. CONCLUSION: The new open reading frame orf1287, representing a chimeric atp6 gene, may play a key role in MAX1 CMS phenotype formation in sunflower, while the contribution of other mitochondrial reorganizations seems to appear negligible for the CMS development.

Probiotic Intake Increases the Expression of Vitellogenin Genes in Laying Hens.

A promising approach for slowing down the rate of reproductive aging is the use of probiotic bacteria as a feed additive. In the current study was investigated the influence of the intake of a potential probiotic on the follicle content and expression of vitellogenin genes (vtg1, vtg2, vtg3) in aged hens. RNA was isolated from liver samples collected from 570-day-old laying hens and gene expression levels were measured using RT-PCR. Bacillus subtilis KATMIRA1933 supplementation had a positive effect on the number of formed follicles in hens and also triggered a significant increase in the relative expression levels of vtg1, vtg2, and vtg3. A Bacillus amyloliquefaciens B-1895 enriched diet or a combination of the two strains had a modest effect on both the number of follicles and the expression of vitellogenin genes. Additionally, the study demonstrates that vitellogenin mRNA expression levels can be considered as a biomarker in a convenient approach for analyzing the hen's egg-laying ability.

Mitochondrial genomes organization in alloplasmic lines of sunflower (Helianthus annuus L.) with various types of cytoplasmic male sterility.

BACKGROUND: Cytoplasmic male sterility (CMS) is a common phenotype in higher plants, that is often associated with rearrangements in mitochondrial DNA (mtDNA), and is widely used to produce hybrid seeds in a variety of valuable crop species. Investigation of the CMS phenomenon promotes understanding of fundamental issues of nuclear-cytoplasmic interactions in the ontogeny of higher plants. In the present study, we analyzed the structural changes in mitochondrial genomes of three alloplasmic lines of sunflower (Helianthus annuus L.). The investigation was focused on CMS line PET2, as there are very few reports about its mtDNA organization. METHODS: The NGS sequencing, de novo assembly, and annotation of sunflower mitochondrial genomes were performed. The comparative analysis of mtDNA of HA89 fertile line and two HA89 CMS lines (PET1, PET2) occurred. RESULTS: The mtDNA of the HA89 fertile line was almost identical to the HA412 line (NC_023337). The comparative analysis of HA89 fertile and CMS (PET1) analog mitochondrial genomes revealed 11,852 bp inversion, 4,732 bp insertion, 451 bp deletion and 18 variant sites. In the mtDNA of HA89 (PET2) CMS line we determined 27.5 kb and 106.5 kb translocations, 711 bp and 3,780 bp deletions, as well as, 5,050 bp and 15,885 bp insertions. There are also 83 polymorphic sites in the PET2 mitochondrial genome, as compared with the fertile line. DISCUSSION: The observed mitochondrial reorganizations in PET1 resulted in only one new open reading frame formation (orfH522), and PET2 mtDNA rearrangements led to the elimination of orf777, duplication of atp6 gene and appearance of four new ORFs with transcription activity specific for the HA89 (PET2) CMS line-orf645, orf2565, orf228 and orf285. Orf228 and orf285 are the atp9 chimeric ORFs, containing transmembrane domains and possibly may impact on mitochondrial membrane potential. So orf228 and orf285 may be the cause for the appearance of the PET2 CMS phenotype, while the contribution of other mtDNA reorganizations in CMS formation is negligible.