Cytotoxic Cyclolignans Obtained by the Enlargement of the Cyclolignan Skeleton of Podophyllic Aldehyde, a Selective Podophyllotoxin-Derived Cyclolignan.

Podophyllotoxin, a cyclolignan natural product, has been the object of extensive chemomodulation to obtain better chemotherapeutic agents. Among the obtained podophyllotoxin derivatives, podophyllic aldehyde showed very interesting potency and selectivity against several tumoral cell lines, so it became our lead compound for further modifications, as described in this work, oriented toward the enlargement of the cyclolignan skeleton. Thus, modifications performed at the aldehyde function included nucleophilic addition reactions and the incorporation of the aldehyde carbon into several five-membered rings, such as thiazolidinones and benzo-fused azoles. The synthesized derivatives were evaluated against several types of cancer cells, and although some compounds were cytotoxic at the nanomolar range, most of them were less potent and less selective than the parent compound podophyllic aldehyde, with the most potent being those having the lactone ring of podophyllotoxin. In silico ADME evaluation predicted good druggability for most of them. The results indicate that the γ-lactone ring is important for potency, while the α,ß-unsaturated aldehyde is necessary to induce selectivity in these cyclolignans.

Non-small cell lung cancer diagnosis aid with histopathological images using Explainable Deep Learning techniques.

BACKGROUND: Lung cancer has the highest mortality rate in the world, twice as high as the second highest. On the other hand, pathologists are overworked and this is detrimental to the time spent on each patient, diagnostic turnaround time, and their success rate. OBJECTIVE: In this work, we design, implement, and evaluate a diagnostic aid system for non-small cell lung cancer detection, using Deep Learning techniques. METHODS: The classifier developed is based on Artificial Intelligence techniques, obtaining an automatic classification result between healthy, adenocarcinoma and squamous cell carcinoma, given an histopathological image from lung tissue. Moreover, a report module based on Explainable Deep Learning techniques is included and gives the pathologist information about the image's areas used to classify the sample and the confidence of belonging to each class. RESULTS: The results show a system accuracy between 97.11 and 99.69%, depending on the number of classes classified, and a value of the area under ROC curve between 99.77 and 99.94%. CONCLUSIONS: The classification results obtain a substantial improvement according to previous works. Thanks to the given report, the time spent by the pathologist and the diagnostic turnaround time can be reduced.

New Antineoplastic Naphthohydroquinones Attached to Labdane and Rearranged Diterpene Skeletons.

Terpenylquinones are mixed biogenesis primary or secondary metabolites widespread in Nature with many biological activities, including the antineoplastic cytotoxicity, that have inspired this work. Here, we present a cytotoxic structure-activity relationship of several diterpenylhydroquinone (DTHQ) derivatives, obtained from the natural labdane diterpenoid myrceocommunic acid used as starting material. Different structural modifications, that changed the functionality and stereochemistry of the decalin, have been implemented on the bicyclic core through epoxidation, ozonolysis or decarboxylation, and through induction of biomimetic breaks and rearrangements of the diterpene skeleton. All the isomers generated were completely characterized by spectroscopic procedures. The resulting compounds have been tested in vitro on cultured cancer cells, showing their relevant antineoplastic cytotoxicity, with GI50 values in the µM and sub-µM range. The rearranged compound 8 showed the best cytotoxic results, with GI50 at the submicromolar range, retaining the cytotoxicity level of the parent compounds. In this report, the versatility of the labdane skeleton for chemical transformation and the interest to continue using structural modifications to obtain new bioactive compounds are demonstrated.

Post-transcriptional Regulation of FLOWERING LOCUS T Modulates Heat-Dependent Source-Sink Development in Potato.

Understanding tuberization in the major crop plant potato (Solanum tuberosum L.) is of importance to secure yield even under changing environmental conditions. Tuber formation is controlled by a homolog of the floral inductor FLOWERING LOCUS T, referred to as SP6A. To gain deeper insights into its function, we created transgenic potato plants overexpressing a codon-optimized version of SP6A, SP6Acop, to avoid silencing effects. These plants exhibited extremely early tuberization at the juvenile stage, hindering green biomass development and indicating a tremendous shift in the source sink balance. The meristem identity was altered in dormant buds of transgenic tubers. This strong phenotype, not being reported so far for plants overexpressing an unmodified SP6A, could be due to post-transcriptional regulation. In fact, a putative SP6A-specific small regulatory RNA was identified in potato. It was effectively repressing SP6A mRNA accumulation in transient assays as well as in leaves of young potato plants prior to tuber formation. SP6A expression is downregulated under heat, preventing tuberization. The molecular mechanism has not been elucidated yet. We showed that this small RNA is strongly upregulated under heat. The importance of the small RNA was demonstrated by overexpression of a target mimicry construct, which led to an increased SP6A expression, enabling tuberization even under continuous heat conditions, which abolished tuber formation in the wild-type. Thus, our study describes an additional regulatory mechanism for SP6A besides the well-known pathway that integrates both developmental and environmental signals to control tuberization and is therefore a promising target for breeding of heat-tolerant potato.

New Hybrids Derived from Podophyllic Aldehyde and Diterpenylhydroquinones with Selectivity toward Osteosarcoma Cells.

A new family of molecular hybrids, between cyclolignans related to podophyllic aldehyde and several diterpenylnaphthohydroquinones (DNHQ), was prepared and its biological activity evaluated in several human solid tumor cell lines, which are representative of the most prevalent solid tumors in the Western world. Both cyclolignan and quinone fragments were linked through aliphatic or aromatic spacers. The new hybrid family was evaluated for its cytotoxicity, and it was found that the hybrids were several times more potent against the osteosarcoma cell line MG-63 than against MCF-7 and HT-29 cell lines. The presence of an aromatic ring in the linker gave the most potent and selective agent, improving the cytotoxicity of the parent compounds. Cell cycle studies demonstrated that this hybrid induces a strong and rapid apoptotic effect and arrests cells at the G2/M phase of the cell cycle, in the same way that the parent compound podophyllic aldehyde does.

Hybrid apomicts trapped in the ecological niches of their sexual ancestors.

Asexual reproduction is expected to reduce the adaptive potential to novel or changing environmental conditions, restricting or altering the ecological niche of asexual lineages. Asexual lineages of plants and animals are typically polyploid, an attribute that may influence their genetic variation, plasticity, adaptive potential, and niche breadth. The genus Boechera (Brassicaceae) represents an ideal model to test the relative ecological and biogeographic impacts of reproductive mode and ploidy because it is composed of diploid sexual and both diploid and polyploid asexual (i.e., apomictic) lineages. Here, we demonstrate a strong association between a transcriptionally conserved allele and apomictic seed formation. We then use this allele as a proxy apomixis marker in 1,649 accessions to demonstrate that apomixis is likely to be a common feature across the Boechera phylogeny. Phylogeographic analyses of these data demonstrate (i) species-specific niche differentiation in sexuals, (ii) extensive niche conservation between differing reproductive modes of the same species, (iii) ploidy-specific niche differentiation within and among species, and (iv) occasional niche drift between apomicts and their sexual ancestors. We conclude that ploidy is a substantially stronger and more common driver of niche divergence within and across Boechera species although variation in both traits may not necessarily lead to niche evolution on the species scale.

Biogeographic variation in genetic variability, apomixis expression and ploidy of St. John's wort (Hypericum perforatum) across its native and introduced range.

BACKGROUND AND AIMS: St. John's wort (Hypericum perforatum) is becoming an important model plant system for investigations into ecology, reproductive biology and pharmacology. This study investigates biogeographic variation for population genetic structure and reproduction in its ancestral (European) and introduced (North America) ranges. METHODS: Over 2000 individuals from 43 localities were analysed for ploidy, microsatellite variation (19 loci) and reproduction (flow cytometric seed screen). Most individuals were tetraploid (93%), while lower frequencies of hexaploid (6%), diploid (<1%) and triploid (<1%) individuals were also identified. KEY RESULTS: A flow cytometric analysis of 24 single seeds per individual, and five individuals per population demonstrated opposite patterns between ploidy types, with tetraploids producing more apomictic (73%) than sexual (24%) seed, while hexaploids produced more sexual (73%) than apomictic (23%) seed. As hexaploids are derived from tetraploids, these data imply that gene dosage, in addition to the effects of hybridization, influences the switch from apomictic to sexual reproduction. No significant differences in seed production were found between Europe and North America. An analysis of population structure based upon microsatellite profiling demonstrated three major genetic clusters in Europe, whose distribution was reflective of Pleistocene glaciation (e.g. refugia) and post-glacial recolonization of Europe. CONCLUSIONS: The presence of pure and mixed populations representing all three genetic clusters in North America demonstrates that H. perforatum was introduced multiple times onto the continent, followed by gene flow between the different gene pools. Taken together, the data presented here suggest that plasticity in reproduction has no influence on the invasive potential of H. perforatum.

A conserved apomixis-specific polymorphism is correlated with exclusive exonuclease expression in premeiotic ovules of apomictic boechera species.

Apomixis (asexual seed production) is characterized by meiotically unreduced egg cell production (apomeiosis) followed by its parthenogenetic development into offspring that are genetic clones of the mother plant. Fertilization (i.e. pseudogamy) of the central cell is important for the production of a functional endosperm with a balanced 2:1 maternal:paternal genome ratio. Here, we present the APOLLO (for apomixis-linked locus) gene, an Aspartate Glutamate Aspartate Aspartate histidine exonuclease whose transcripts are down-regulated in sexual ovules entering meiosis while being up-regulated in apomeiotic ovules at the same stage of development in plants of the genus Boechera. APOLLO has both "apoalleles," which are characterized by a set of linked apomixis-specific polymorphisms, and "sexalleles." All apomictic Boechera spp. accessions proved to be heterozygous for the APOLLO gene (having at least one apoallele and one sexallele), while all sexual genotypes were homozygous for sexalleles. Apoalleles contained a 20-nucleotide polymorphism present in the 5' untranslated region that contains specific transcription factor-binding sites for ARABIDOPSIS THALIANA HOMEOBOX PROTEIN5, LIM1 (for LINEAGE ABNORMAL11, INSULIN1, MECHANOSENSORY PROTEIN3), SORLIP1AT (for SEQUENCES OVERREPRESENTED IN LIGHT-INDUCED PROMOTERS IN ARABIDOPSIS THALIANA1), SORLIP2AT, and POLYA SIGNAL1. In the same region, sexalleles contain transcription factor-binding sites for DNA BINDING WITH ONE FINGER2, DNA BINDING WITH ONE FINGER3, and PROLAMIN BOX-BINDING FACTOR. Our results suggest that the expression of a single deregulated allele could induce the cascade of events leading to asexual female gamete formation in an apomictic plant.

Synthesis and evaluation as antitumor agents of 1,4-naphthohydroquinone derivatives conjugated with amino acids and purines.

We report on the synthesis of two series of 1,4-naphthohydroquinone derivatives conjugated with amino acids (Gly, Ala, Phe, and Glu) and with substituted purines linked by an aliphatic chain. The compounds were obtained through Diels-Alder cycloaddition between myrcene and 1,4-benzoquinone and evaluated in vitro for their cytotoxicity (GI50 ) against cultured human cancer cells of A-549 lung carcinoma, HT-29 colon adenocarcinoma, and MCF-7 breast carcinoma. The GI50 values found for some hydroquinone-amino acid and hydroquinone-purine hybrids against MCF-7 are in an activity range comparable to that of the reference drug doxorubicin.

The conserved chimeric transcript UPGRADE2 is associated with unreduced pollen formation and is exclusively found in apomictic Boechera species.

In apomictic Boechera spp., meiotic diplospory leads to the circumvention of meiosis and the suppression of recombination to produce unreduced male and female gametes (i.e. apomeiosis). Here, we have established an early flower developmental staging system and have performed microarray-based comparative gene expression analyses of the pollen mother cell stage in seven diploid sexual and seven diploid apomictic genotypes to identify candidate factors for unreduced pollen formation. We identified a transcript unique to apomictic Boechera spp. called UPGRADE2 (BspUPG2), which is highly up-regulated in their pollen mother cells. BspUPG2 is highly conserved among apomictic Boechera spp. genotypes but has no homolog in sexual Boechera spp. or in any other taxa. BspUPG2 undergoes posttranscriptional processing but lacks a prominent open reading frame. Together with the potential of stably forming microRNA-like secondary structures, we hypothesize that BspUPG2 functions as a long regulatory noncoding messenger RNA-like RNA. BspUPG2 has apparently arisen through a three-step process initiated by ancestral gene duplication of the original BspUPG1 locus, followed by sequential insertions of segmentally duplicated gene fragments, with final exonization of its sequence structure. Its genesis reflects the hybridization history that characterizes the genus Boechera.

Copy number variation in transcriptionally active regions of sexual and apomictic Boechera demonstrates independently derived apomictic lineages.

In asexual (apomictic) plants, the absence of meiosis and sex is expected to lead to mutation accumulation. To compare mutation accumulation in the transcribed genomic regions of sexual and apomictic plants, we performed a double-validated analysis of copy number variation (CNV) on 10 biological replicates each of diploid sexual and diploid apomictic Boechera, using a high-density (>700 K) custom microarray. The Boechera genome demonstrated higher levels of depleted CNV, compared with enriched CNV, irrespective of reproductive mode. Genome-wide patterns of CNV revealed four divergent lineages, three of which contain both sexual and apomictic genotypes. Hence genome-wide CNV reflects at least three independent origins (i.e., expression) of apomixis from different sexual genetic backgrounds. CNV distributions for different families of transposable elements were lineage specific, and the enrichment of LINE/L1 and long term repeat/Copia elements in lineage 3 apomicts is consistent with sex and meiosis being mechanisms for purging genomic parasites. We hypothesize that significant overrepresentation of specific gene ontology classes (e.g., pollen-pistil interaction) in apomicts implies that gene enrichment could be an adaptive mechanism for genome stability in diploid apomicts by providing a polyploid-like system for buffering the effects of deleterious mutations.

Synthesis, cytotoxicity and antiplasmodial activity of novel ent-kaurane derivatives.

This paper reports on the syntheses and spectrometric characterisation of eleven novel ent-kaurane diterpenoids, including a complete set of (1)H, (13)C NMR and crystallographic data for two novel ent-kaurane diepoxides. Moreover, the antineoplastic cytotoxicity for kaurenoic acid and the majority of ent-kaurane derivatives were assessed in vitro against a panel of fourteen cancer cell lines, of which allylic alcohols were shown to be the most active compounds. The good in vitro antimalarial activity and the higher selectivity index values observed for some ent-kaurane epoxides against the chloroquine-resistant W2 clone of Plasmodium falciparum indicate that this class of natural products may provide new hits for the development of antimalarial drugs.

Novel microRNAs and microsatellite-like small RNAs in sexual and apomictic Boechera species.

Apomixis refers to plant asexual reproduction through seeds that give rise to progeny which are genotypically identical to the maternal parent. It has evolved from many different sexual taxa although the underlying genetic factors remain unknown. Previous analyses of the over-representation of transcription factors, in a comparison of microdissected ovules from apomictic and sexual Boechera, showed that many transcription factor mRNAs possessed microRNA (miRNAs) binding sites, thus pointing to miRNAs as potentially important factors that may be involved in the regulatory switch from sexual to apomictic reproduction. A microarray-based approach was used to identify (1) 673 microsatellitelike small RNAs (misRNAs) containing predominantly 2-7 repeats of (GAA)n/(CUU)n, (GCA)n/(CGU)n, (GGA)n/(CCU)n, (GGU)n/(CCA)n and (UGA)n/(ACU)n, and (2) 166 more typical non-repeat small RNAs. In total, 87 small RNAs were found to be located in cDNAs that could fold into stem-loop structures and thus represent miRNA molecules. In addition, 109 Boechera small RNAs including both misRNAs and non-repeat small RNAs, showed significant homology to 407 Arabidopsis thaliana small RNAs including the A. thaliana pollen-specific ath-miR5021. This indicates that only a fraction of the identified small RNAs are unique to Boechera. Ten small RNAs were validated using a Northern blot assay on flower and leaf tissues, eight of which showed flower-specific expression with varying abundance. The potential binding sites of many of the misRNAs and non-repeat small RNAs occur predominantly in exonic regions. This feature coupled with their flower-specific pattern of expression is suggestive of their probable role in post-transcriptional gene regulation. We propose that quantitative variation for misRNA target binding (and hence post-transcriptional gene regulation) could arise via microsatellite length polymorphisms occurring either in misRNA precursors or in their gene targets.

New naphthoquinones and an alkaloid with in vitro activity against Toxoplasma gondii RH and EGS strains.

The efficacy of three amino-terpenyl naphthoquinones and the alkaloid liriodenine were examined against tachyzoites and tissues cysts of the RH and EGS strains, respectively. Monolayers of 2C4 fibroblasts infected with tachyzoites of the RH strain were incubated with different concentrations of the compounds for 48 h. Specifically, 7-(4-methyl-3-pentenyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-5), 6-(4-methyl-3-pentenyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-6), 6-(4-methylpentyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-11), and 8 h-benzo[g]-1,3-benzodioxolo[6,5,4-de]quinolin-8-one,9Cl-1,2-methylene dioxiaporfina (liriodenine) inhibited intracellular replication of T. gondii. The IC(50) values obtained for compounds QUI-5 and QUI-6 were 69.35 and 172.81 µM (i.e., 21.4 and 53.4 µg/mL), respectively. The naphthoquinone QUI-11 and liriodenine significantly inhibited intracellular replication of T. gondii. The IC(50) values obtained with these experiments were 0.32 and 0.07 µM (i.e., 0.1 and 0.02 µg/mL), respectively. Compounds QUI-5, QUI-6, QUI-11 and liriodenine demonstrated lower toxicity for 2C4 fibroblasts compared to atovaquone. In addition, cysts isolated from the brains of mice chronically infected with the EGS strain were exposed to the compounds. Infectivity of the cysts after incubation with the compounds was assessed by infection of mice. The data obtained showed that in vitro incubation with QUI-6, QUI-11 and liriodenine inhibited the infectivity of the bradyzoites. This activity was time- and concentration-dependent.

Analysis of conserved microRNAs in floral tissues of sexual and apomictic Boechera species.

BACKGROUND: Apomixis or asexual seed formation represents a potentially important agronomic trait whose introduction into crop plants could be an effective way to fix and perpetuate a desirable genotype through successive seed generations. However, the gene regulatory pathways underlying apomixis remain unknown. In particular, the potential function of microRNAs, which are known to play crucial roles in many aspects of plant growth and development, remains to be determined with regards to the switch from sexual to apomictic reproduction. RESULTS: Using bioinformatics and microarray validation procedures, 51 miRNA families conserved among angiosperms were identified in Boechera. Microarray assay confirmed 15 of the miRNA families that were identified by bioinformatics techniques. 30 cDNA sequences representing 26 miRNAs could fold back into stable pre-miRNAs. 19 of these pre-miRNAs had miRNAs with Boechera-specific nucleotide substitutions (NSs). Analysis of the Gibbs free energy (ΔG) of these pre-miRNA stem-loops with NSs showed that the Boechera-specific miRNA NSs significantly (p ≤ 0.05) enhance the stability of stem-loops. Furthermore, six transcription factors, the Squamosa promoter binding protein like SPL6, SPL11 and SPL15, Myb domain protein 120 (MYB120), RELATED TO AP2.7 DNA binding (RAP2.7, TOE1 RAP2.7) and TCP family transcription factor 10 (TCP10) were found to be expressed in sexual or apomictic ovules. However, only SPL11 showed differential expression with significant (p ≤ 0.05) up-regulation at the megaspore mother cell (MMC) stage of ovule development in apomictic genotypes. CONCLUSIONS: This study constitutes the first extensive insight into the conservation and expression of microRNAs in Boechera sexual and apomictic species. The miR156/157 target squamosa promoter binding protein-like 11 (SPL11) was found differentially expressed with significant (p ≤ 0.05) up-regulation at the MMC stage of ovule development in apomictic genotypes. The results also demonstrate that nucleotide changes in mature miRNAs significantly (p ≤ 0.05) enhance the thermodynamic stability of pre-miRNA stem-loops.

Isolation and characterization of microsatellite loci from apomictic Hypericum perforatum (Hypericaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed to study the origins and evolution of apomictic Hypericum perforatum, a polyploid invasive perennial herb indigenous to Europe that produces compounds of medicinal/pharmaceutical importance. METHODS AND RESULTS: Eleven primer sets were developed using a non-radioactive protocol. All loci were polymorphic, showing from 2 to 9 alleles per locus. Observed and expected heterozygosity averaged 0.861 and 0.772, respectively, across four studied populations. Most primers also amplified successfully in eight other Hypericum species. CONCLUSIONS: Markers developed in this study provide genetic tools for studies of apomixis and invasiveness, both on the intra- and interspecific levels.

iso-Kaurenoic acid from Wedelia paludosa D.C.

A recent reinvestigation of aerial parts of Wedelia paludosa D.C. is described and reports, for the first time, the isolation of iso-kaurenoic acid from this species.

iso-Kaurenoic acid from Wedelia paludosa D.C.

A recent reinvestigation of aerial parts of Wedelia paludosa D.C. is described and reports, for the first time, the isolation of iso-kaurenoic acid from this species.

Uma recente reinvestigação das partes aéreas de Wedelia paludosa D.C. é descrita e relata, pela primeira vez, o isolamento do ácido iso-caurenóico desta espécie.

Apomictic and sexual ovules of Boechera display heterochronic global gene expression patterns.

We have compared the transcriptomic profiles of microdissected live ovules at four developmental stages between a diploid sexual and diploid apomictic Boechera. We sequenced >2 million SuperSAGE tags and identified (1) heterochronic tags (n = 595) that demonstrated significantly different patterns of expression between sexual and apomictic ovules across all developmental stages, (2) stage-specific tags (n = 577) that were found in a single developmental stage and differentially expressed between the sexual and apomictic ovules, and (3) sex-specific (n = 237) and apomixis-specific (n = 1106) tags that were found in all four developmental stages but in only one reproductive mode. Most heterochronic and stage-specific tags were significantly downregulated during early apomictic ovule development, and 110 were associated with reproduction. By contrast, most late stage-specific tags were upregulated in the apomictic ovules, likely the result of increased gene copy number in apomictic (hexaploid) versus sexual (triploid) endosperm or of parthenogenesis. Finally, we show that apomixis-specific gene expression is characterized by a significant overrepresentation of transcription factor activity. We hypothesize that apomeiosis is associated with global downregulation at the megaspore mother cell stage. As the diploid apomict analyzed here is an ancient hybrid, these data are consistent with the postulated link between hybridization and asexuality and provide a hypothesis for multiple evolutionary origins of apomixis in the genus Boechera.

Synthesis and cytotoxic evaluation of 6-(3-pyrazolylpropyl) derivatives of 1,4-naphthohydroquinone-1,4-diacetate.

Several new 6-(3-pyrazolylpropyl) derivatives of 1,4-naphthohydroquinone-1,4-diacetate (NHQ-DA) have been prepared by chemical modifications of the Diels-Alder adduct of alpha-myrcene and 1,4-benzoquinone. All these new compounds and precursors have been evaluated in vitro for their cytotoxicity against cultured human cancer cells of MB-231 breast-adeno carcinoma, A-549 lung carcinoma, and HT-29 colon carcinoma. GI(50) values ranged in and below the micromolar concentration level.