High resolution mapping of Twist to DNA in Drosophila embryos: Efficient functional analysis and evolutionary conservation

Cis-regulatory modules (CRMs) function by binding sequence specific transcription factors, but the relationship between in vivo physical binding and the regulatory capacity of factor-bound DNA elements remains uncertain. We investigate this relationship for the well-studied Twist factor in Drosophila melanogaster embryos by analyzing genome-wide factor occupancy and testing the functional significance of Twist occupied regions and motifs within regions. Twist ChIP-seq data efficiently identified previously studied Twist-dependent CRMs and robustly predicted new CRM activity in transgenesis, with newly identified Twist-occupied regions supporting diverse spatiotemporal patterns (>74% positive, n = 31). Some, but not all, candidate CRMs require Twist for proper expression in the embryo. The Twist motifs most favored in genome ChIP data (in vivo) differed from those most favored by Systematic Evolution of Ligands by EXponential enrichment (SELEX) (in vitro). Furthermore, the majority of ChIP-seq signals could be parsimoniously explained by a CABVTG motif located within 50 bp of the ChIP summit and, of these, CACATG was most prevalent. Mutagenesis experiments demonstrated that different Twist E-box motif types are not fully interchangeable, suggesting that the ChIP-derived consensus (CABVTG) includes sites having distinct regulatory outputs. Further analysis of position, frequency of occurrence, and sequence conservation revealed significant enrichment and conservation of CABVTG E-box motifs near Twist ChIP-seq signal summits, preferential conservation of ±150 bp surrounding Twist occupied summits, and enrichment of GA- and CA-repeat sequences near Twist occupied summits. Our results show that high resolution in vivo occupancy data can be used to drive efficient discovery and dissection of global and local cis-regulatory logic

Comparative Analysis of Plastid Genomes Using Pangenome Research ToolKit (PGR-TK)

Plastid genomes (plastomes) of angiosperms are of great interest among biologists. High-throughput sequencing is making many such genomes accessible, increasing the need for tools to perform rapid comparative analysis. This exploratory analysis investigates whether the Pangenome Research Tool Kit (PGR-TK) is suitable for analyzing plastomes. After determining the optimal parameters for this tool on plastomes, we use it to compare sequences from each of the genera - Magnolia, Solanum, Fragaria and Cotoneaster, as well as a combined set from 20 rosid genera. PGR-TK recognizes large-scale plastome structures, such as the inverted repeats, among combined sequences from distant rosid families. If the plastid genomes are rotated to the same starting point, it also correctly groups different species from the same genus together in a generated cladogram. The visual approach of PGR-TK provides insights into genome evolution without requiring gene annotations.Comment: 15 pages, 4 figure

Analysis of Antisense Expression by Whole Genome Tiling Microarrays and siRNAs Suggests Mis-Annotation of Arabidopsis Orphan Protein-Coding Genes

MicroRNAs (miRNAs) and trans-acting small-interfering RNAs (tasi-RNAs) are small (20-22 nt long) RNAs (smRNAs) generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs) are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery.We explored rice (Oryza sativa) sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans) and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis 'orphan' hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM) was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the "ancient" (deeply conserved) class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for "new" rapidly-evolving MIRNA genes.Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non-coding RNAs in plants and potentially other kingdoms, which can provide insight into antisense transcription, miRNA evolution, and post-transcriptional gene regulation

A Thiazole Coumarin (TC) turn-on fluorescence probe for AT-base pair detection and multipurpose applications in different biological systems

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based Thiazole Coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-Activated Cell Sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology

Computational and transcriptional evidence for microRNAs in the honey bee genome

A total of 68 non-redundant candidate honey bee miRNAs were identified computationally; several of them appear to have previously unrecognized orthologs in the Drosophila genome. Several miRNAs showed caste- or age-related differences in transcript abundance and are likely to be involved in regulating honey bee development

Traditional uses, phytochemistry, pharmacology and toxicology of garlic (Allium sativum), a storehouse of diverse phytochemicals: A review of research from the last decade focusing on health and nutritional implications

Allium sativum L. (Garlic) is a fragrant herb and tuber-derived spice that is one of the most sought-after botanicals, used as a culinary and ethnomedicine for a variety of diseases around the world. An array of pharmacological attributes such as antioxidant, hypoglycemic, anti-inflammatory, antihyperlipidemic, anticancer, antimicrobial, and hepatoprotective activities of this species have been established by previous studies. A. sativum houses many sulfur-containing phytochemical compounds such as allicin, diallyl disulfide (DADS), vinyldithiins, ajoenes (E-ajoene, Z-ajoene), diallyl trisulfide (DATS), micronutrient selenium (Se) etc. Organosulfur compounds are correlated with modulations in its antioxidant properties. The garlic compounds have also been recorded as promising immune-boosters or act as potent immunostimulants. A. sativum helps to treat cardiovascular ailments, neoplastic growth, rheumatism, diabetes, intestinal worms, flatulence, colic, dysentery, liver diseases, facial paralysis, tuberculosis, bronchitis, high blood pressure, and several other diseases. The present review aims to comprehensively enumerate the ethnobotanical and pharmacological aspects of A. sativum with notes on its phytochemistry, ethnopharmacology, toxicological aspects, and clinical studies from the retrieved literature from the last decade with notes on recent breakthroughs and bottlenecks. Future directions related to garlic research is also discussed.This work was supported by the UHK (Project No. VT2019- 2021), APOGEO (Cooperation Program INTERREG-MAC 2014–2020), with European Funds for Regional Development- FEDER, the “Agencia Canaria de Investigación, Innovación y Sociedad de la Información (ACIISI) Gobierno de Canarias” (Project No. ProID2020010134), and Caja Canarias (Project No. 2019SP43).Peer reviewe