Verbascum species as a new source of saffron apocarotenoids and molecular tools for the biotechnological production of crocins and picrocrocin.

Crocins are glucosylated apocarotenoids present in flowers and fruits of a few plant species, including saffron, gardenia, and Buddleja. The biosynthesis of crocins in these plants has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems. Mullein (Verbascum sp.) has been identified as a new source of crocins and picrocrocin. In this work, we have identified eight enzymes involved in the cleavage of carotenoids in two Verbascum species, V. giganteum and V. sinuatum. Four of them were homologous to the previously identified BdCCD4.1 and BdCCD4.3 from Buddleja, involved in the biosynthesis of crocins. These enzymes were analyzed for apocarotenogenic activity in bacteria and Nicotiana benthamiana plants using a virus-driven system. Metabolic analyses of bacterial extracts and N. benthamiana leaves showed the efficient activity of these enzymes to produce crocins using ß-carotene and zeaxanthin as substrates. Accumulations of 0.17% of crocins in N. benthamiana dry leaves were reached in only 2 weeks using a recombinant virus expressing VgCCD4.1, similar to the amounts previously produced using the canonical saffron CsCCD2L. The identification of these enzymes, which display a particularly broad substrate spectrum, opens new avenues for apocarotenoid biotechnological production.

A novel conservative technique to allow oral opening and overcome rigor mortis during medicolegal autopsy.

This study presents a novel conservative technique to increase oral opening and overcome rigor mortis during medicolegal autopsy (MLA). The method proposes a myotomy of the temporalis and masseter muscles, taking advantage of the incisions of the cranial opening procedures, to achieve a significant oral opening (≥ 30 mm) in fresh cadavers with established rigor mortis without altering facial aesthetics. The study was performed on 48 individuals aged between 18 and 66 years (20 males and 28 females) who were autopsied at the Instituto Nacional de Patología Forense Dr. Sergio Sarita Valdez (Santo Domingo, Dominican Republic). The proposed technique not only allowed the oral access to all autopsied cadavers but also complied with current ethical standards for preserving facial aesthetics, taking into account the concerns of family members by allowing respectful viewing of the cadaver. This advancement not only contributes to the efficiency of oral autopsies but also influences public perception of forensic procedures and highlights the importance of ethical and aesthetic considerations in the context of MLAs. The proposed technique represents a significant step towards more respectful and ethical forensic practices, and offers practical advantages for a more complete and accurate assessment during MLA.

Thymoquinone-Loaded Chitosan Nanoparticles as Natural Preservative Agent in Cosmetic Products.

The current status of controversy regarding the use of certain preservatives in cosmetic products makes it necessary to seek new ecological alternatives that are free of adverse effects on users. In our study, the natural terpene thymoquinone was encapsulated in chitosan nanoparticles. The nanoparticles were characterized by DLS and TEM, showing a particle size of 20 nm. The chemical structure, thermal properties, and release profile of thymoquinone were evaluated and showed a successful stabilization and sustained release of terpenes. The antimicrobial properties of the nanoparticles were evaluated against typical microbial contaminants found in cosmetic products, showing high antimicrobial properties. Furthermore, natural moisturizing cream inoculated with the aforementioned microorganisms was formulated with thymoquinone-chitosan nanoparticles to evaluate the preservative efficiency, indicating its promising use as a preservative in cosmetics.

Subfunctionalization of D27 Isomerase Genes in Saffron.

Chromoplasts and chloroplasts contain carotenoid pigments as all-trans- and cis-isomers, which function as accessory light-harvesting pigments, antioxidant and photoprotective agents, and precursors of signaling molecules and plant hormones. The carotenoid pathway involves the participation of different carotenoid isomerases. Among them, D27 is a ß-carotene isomerase showing high specificity for the C9-C10 double bond catalyzing the interconversion of all-trans- into 9-cis-ß-carotene, the precursor of strigolactones. We have identified one D27 (CsD27-1) and two D27-like (CsD27-2 and CsD27-3) genes in saffron, with CsD27-1 and CsD27-3, clearly differing in their expression patterns; specifically, CsD27-1 was mainly expressed in the undeveloped stigma and roots, where it is induced by Rhizobium colonization. On the contrary, CsD27-2 and CsD27-3 were mainly expressed in leaves, with a preferential expression of CsD27-3 in this tissue. In vivo assays show that CsD27-1 catalyzes the isomerization of all-trans- to 9-cis-ß-carotene, and could be involved in the isomerization of zeaxanthin, while CsD27-3 catalyzes the isomerization of all-trans- to cis-ζ-carotene and all-trans- to cis-neurosporene. Our data show that CsD27-1 and CsD27-3 enzymes are both involved in carotenoid isomerization, with CsD27-1 being specific to chromoplast/amyloplast-containing tissue, and CsD27-3 more specific to chloroplast-containing tissues. Additionally, we show that CsD27-1 is co-expressed with CCD7 and CCD8 mycorrhized roots, whereas CsD27-3 is expressed at higher levels than CRTISO and Z-ISO and showed circadian regulation in leaves. Overall, our data extend the knowledge about carotenoid isomerization and their implications in several physiological and ecological processes.

Current lifestyle and exclusive breastfeeding: an impossible balance?

427 women were included in this observational cohort study to determine the rate of exclusive (EB), mixed (MB) and artificial breastfeeding (AB) and the personal reasons and social factors influencing this decision. An initial survey was conducted within the first 36 h postpartum and a second wave was carried out 3 months after delivery. 55.74% (238) of the patients intended to EB, 32.55% (139) AB and 11.71% (50) MB. After 3 months, a high percentage in group EB (75.23%) maintained EB. The main reason for switching to AB was the absence of milk or weight loss of the newborn. In conclusion, EB is the preferred form for feeding newborns. When these women returned to their workplace, most of them wanted to continue with EB. Women with higher academic degrees chose more often EB.IMPACT STATEMENTWhat is already known on this subject? The benefits of exclusive breastfeeding are well known. However, in modern western societies, it is not easy to combine breastfeeding with day-to-day activity. Work activity, age or previous parity are some of the factors that may influence the election of the type of lactation.What do the results of this study add? Exclusive breastfeeding is the preferred method for feeding newborns immediately postpartum and 3 months later. Work activity does not seem to influence or to switch the choice. However, the level of education could be a determinant of adherence to exclusive breastfeeding.What are the implications of these findings for clinical practice and/or further research? Assessing the factors that favour adherence to exclusive breastfeeding might be a useful tool to its promotion. This study warrants further multivariate analyses on the same topic and additional studies in other social contexts.

Identification and characterization of apocarotenoid modifiers and carotenogenic enzymes for biosynthesis of crocins in Buddleja davidii flowers.

Crocetin biosynthesis in Buddleja davidii flowers proceeds through a zeaxanthin cleavage pathway catalyzed by two carotenoid cleavage dioxygenases (BdCCD4.1 and BdCCD4.3), followed by oxidation and glucosylation reactions that lead to the production of crocins. We isolated and analyzed the expression of 12 genes from the carotenoid pathway in B. davidii flowers and identified four candidate genes involved in the biosynthesis of crocins (BdALDH, BdUGT74BC1, BdUGT74BC2, and BdUGT94AA3). In addition, we characterized the profile of crocins and their carotenoid precursors, following their accumulation during flower development. Overall, seven different crocins, crocetin, and picrocrocin were identified in this study. The accumulation of these apocarotenoids parallels tissue development, reaching the highest concentration when the flower is fully open. Notably, the pathway was regulated mainly at the transcript level, with expression patterns of a large group of carotenoid precursor and apocarotenoid genes (BdPSY2, BdPDS2, BdZDS, BdLCY2, BdBCH, BdALDH, and BdUGT Genes) mimicking the accumulation of crocins. Finally, we used comparative correlation network analysis to study how the synthesis of these valuable apocarotenoids diverges among B. davidii, Gardenia jasminoides, and Crocus sativus, highlighting distinctive differences which could be the basis of the differential accumulation of crocins in the three species.

A New Glycosyltransferase Enzyme from Family 91, UGT91P3, Is Responsible for the Final Glucosylation Step of Crocins in Saffron (Crocus sativus L.).

Crocetin is an apocarotenoid formed from the oxidative cleavage of zeaxanthin, by the carotenoid cleavage enzymes CCD2 (in Crocus species) and specific CCD4 enzymes in Buddleja davidii and Gardenia jasminoides. Crocetin accumulates in the stigma of saffron in the form of glucosides and crocins, which contain one to five glucose molecules. Crocetin glycosylation was hypothesized to involve at least two enzymes from superfamily 1 UDP-sugar dependent glycosyltransferases. One of them, UGT74AD1, produces crocins with one and two glucose molecules, which are substrates for a second UGT, which could belong to the UGT79, 91, or 94 families. An in silico search of Crocus transcriptomes revealed six candidate UGT genes from family 91. The transcript profiles of one of them, UGT91P3, matched the metabolite profile of crocin accumulation, and were co-expressed with UGT74AD1. In addition, both UGTs interact in a two-hybrid assay. Recombinant UGT91P3 produced mostly crocins with four and five glucose molecules in vitro, and in a combined transient expression assay with CCD2 and UGT74AD1 enzymes in Nicotiana benthamiana. These results suggest a role of UGT91P3 in the biosynthesis of highly glucosylated crocins in saffron, and that it represents the last missing gene in crocins biosynthesis.

Efficient production of saffron crocins and picrocrocin in Nicotiana benthamiana using a virus-driven system.

Crocins and picrocrocin are glycosylated apocarotenoids responsible, respectively, for the color and the unique taste of the saffron spice, known as red gold due to its high price. Several studies have also shown the health-promoting properties of these compounds. However, their high costs hamper the wide use of these metabolites in the pharmaceutical sector. We have developed a virus-driven system to produce remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana plants in only two weeks. The system consists of viral clones derived from tobacco etch potyvirus that express specific carotenoid cleavage dioxygenase (CCD) enzymes from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues demonstrated that the sole virus-driven expression of C. sativus CsCCD2L or B. davidii BdCCD4.1 resulted in the production of crocins, picrocrocin and safranal. Using the recombinant virus that expressed CsCCD2L, accumulations of 0.2% of crocins and 0.8% of picrocrocin in leaf dry weight were reached in only two weeks. In an attempt to improve apocarotenoid content in N. benthamiana, co-expression of CsCCD2L with other carotenogenic enzymes, such as Pantoea ananatis phytoene synthase (PaCrtB) and saffron ß-carotene hydroxylase 2 (BCH2), was performed using the same viral system. This combinatorial approach led to an additional crocin increase up to 0.35% in leaves in which CsCCD2L and PaCrtB were co-expressed. Considering that saffron apocarotenoids are costly harvested from flower stigma once a year, and that Buddleja spp. flowers accumulate lower amounts, this system may be an attractive alternative for the sustainable production of these appreciated metabolites.

An olaparib window-of-opportunity trial in patients with early-stage endometrial carcinoma: POLEN study.

OBJECTIVE: Olaparib is a potent inhibitor of poly(ADP-ribose) polymerase (PARP)-1, 2, and 3 with potential activity in endometrial cancer (EC). METHODS: In this window-of-opportunity trial, women with operable type 1 EC received olaparib oral tablets (300mg) twice daily for 28days before surgery. The primary objective was to evaluate the effects of olaparib on EC in tissue samples taken at baseline and at treatment completion. Signal of activity was defined as significant changes in the expression of the cell cycle-related proteins cyclin D1, Ki67, and cleaved caspase-3. RESULTS: A total of 31 patients were included in the biomarker analysis. The median time of olaparib exposure was 24 days (1-39). Significant inhibition was found for cyclin D1 (p < 0.01), but not for Ki67 and active caspase 3 immunostaining. PARP-1 levels positively correlated with cyclin D1 levels (rho = 0.661, p = 0.0001). Both PARP-1 and cyclin D1 levels were significantly lower (p = 0.022 and p = 0.004, respectively) in patients with ARID1A[-] tumors than ARID1A[+] tumors. A significant relationship between plasma olaparib concentrations and decreased GLUT1 activity was observed (r = -0.5885; p < 0.05). Drug-related toxicity consisted mostly of gastrointestinal and grade 1 or 2 adverse events. CONCLUSIONS: Olaparib reduced expression of cyclin D1, which positively correlated with PARP-1 levels. This effect was more evident in ARID1A-deficient tumors. Olaparib further induced inhibition of GLUT1 plasma activity. Our findings could have noteworthy implications in predicting which patients with EC would benefit from olaparib-based strategies.

Emotional Functioning, Positive Relationships, and Language Use in Deaf Adults.

This study aimed to explore emotional regulation, alexithymia, and interpersonal relations in a Spanish sample of 146 adult deaf participants and 146 typical hearing participants. For the deaf sample, the associations between type of language used and the above variables were also analyzed. Results showed that deaf participants scored higher on alexithymia and lower for positive relationships. However, no differences were found between deaf and typical hearing participants on emotional regulation. Regarding language use (Spanish Oral Language, SOL; Spanish Sign Language; or both), deaf participants used both languages in most contexts. Deaf participants who reported using SOL as their main mode of expression showed greater positive relationships than deaf participants who use both languages. Deaf participants who reported using both languages scored higher in emotional attention and emotional repair. These results evidence an association between the language use of deaf participants and alexithymia.

Multi-species transcriptome analyses for the regulation of crocins biosynthesis in Crocus.

BACKGROUND: Crocins are soluble apocarotenoids that mainly accumulate in the stigma tissue of Crocus sativus and provide the characteristic red color to saffron spice, in addition to being responsible for many of the medicinal properties of saffron. Crocin biosynthesis and accumulation in saffron is developmentally controlled, and the concentration of crocins increases as the stigma develops. Until now, little has been known about the molecular mechanisms governing crocin biosynthesis and accumulation. This study aimed to identify the first set of gene regulatory processes implicated in apocarotenoid biosynthesis and accumulation. RESULTS: A large-scale crocin-mediated RNA-seq analysis was performed on saffron and two other Crocus species at two early developmental stages coincident with the initiation of crocin biosynthesis and accumulation. Pairwise comparison of unigene abundance among the samples identified potential regulatory transcription factors (TFs) involved in crocin biosynthesis and accumulation. We found a total of 131 (up- and downregulated) TFs representing a broad range of TF families in the analyzed transcriptomes; by comparison with the transcriptomes from the same developmental stages from other Crocus species, a total of 11 TF were selected as candidate regulators controlling crocin biosynthesis and accumulation. CONCLUSIONS: Our study generated gene expression profiles of stigmas at two key developmental stages for apocarotenoid accumulation in three different Crocus species. Differential gene expression analyses allowed the identification of transcription factors that provide evidence of environmental and developmental control of the apocarotenoid biosynthetic pathway at the molecular level.

UGT709G1: a novel uridine diphosphate glycosyltransferase involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron (Crocus sativus).

Saffron, a spice derived from the dried red stigmas of Crocus sativus, is one of the oldest natural food additives. The flowers have long red stigmas, which store significant quantities of the glycosylated apocarotenoids crocins and picrocrocin. The apocarotenoid biosynthetic pathway in saffron starts with the oxidative cleavage of zeaxanthin, from which crocins and picrocrocin are derived. In the processed stigmas, picrocrocin is converted to safranal, giving saffron its typical aroma. By a targeted search for differentially expressed uridine diphosphate glycosyltransferases (UGTs) in Crocus transcriptomes, a novel apocarotenoid glucosyltransferase (UGT709G1) from saffron was identified. Biochemical analyses revealed that UGT709G1 showed a high catalytic efficiency toward 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene (HTCC), making it suited for the biosynthesis of picrocrocin, the precursor of safranal. The role of UGT709G1 in picrocrocin/safranal biosynthesis was supported by the absence or presence of gene expression in a screening for HTCC and picrocrocin production in different Crocus species and by a combined transient expression assay with CsCCD2L in Nicotiana benthamiana leaves. The identification of UGT709G1 completes one of the most highly valued specialized metabolic biosynthetic pathways in plants and provides novel perspectives on the industrial production of picrocrocin to be used as a flavor additive or as a pharmacological constituent.

Identification of the cellular components involved in de novo immune hepatitis: a quantitative immunohistochemical analysis.

BACKGROUND: Diagnosis of de novo immune hepatitis (dnIH) after liver transplantation relies on biopsy findings, with an abundance of plasma cells (PCs) in the inflammatory infiltrates a hallmark of the disease. Very little is known about what other types of immune cells exist in the infiltrates mainly located in the portal areas of the liver tissue. METHODS: We analyzed the composition of T cells, B cells, PCs, and macrophages in the liver biopsies of 12 patients with dnIH, 9 of them obtained at the time of diagnosis. For comparison, biopsies from 9 patients with chronic rejection (CR) were included in the study. The results were analyzed by a computer-assisted stereology quantification method. RESULTS: The major components of the infiltrates in the portal areas were CD3+ T lymphocytes in both groups, with 36.6% in the dnIH group versus 49.4% in the CR group. CD20+ B lymphocytes represented 14.9% in the dnIH group and 29.1% in the CR group. Macrophage levels were very similar in the dnIH and CR group (19.7% versus 16.8%, respectively). PCs were much less represented in CR biopsies than those from the dnIH group (mean value of 4.7% versus 28.8%). CONCLUSION: In conclusion, the determination of a characteristic cellular profile could be an important tool for a more reliable diagnosis of dnIH, in support of the histological evaluation made by the pathologist, which in most cases is challenging. Recognition of this condition is crucial because it leads to graft failure if left untreated.

Expression and Interaction Analysis among Saffron ALDHs and Crocetin Dialdehyde.

In saffron, the cleavage of zeaxanthin by means of CCD2 generates crocetin dialdehyde, which is then converted by an unknown aldehyde dehydrogenase to crocetin. A proteome from saffron stigma was released recently and, based on the expression pattern and correlation analyses, five aldehyde dehydrogenases (ALDHs) were suggested as possible candidates to generate crocetin from crocetin dialdehydes. We selected four of the suggested ALDHs and analyzed their expression in different tissues, determined their activity over crocetin dialdehyde, and performed structure modeling and docking calculation to find their specificity. All the ALDHs were able to convert crocetin dialdehyde to crocetin, but two of them were stigma tissue-specific. Structure modeling and docking analyses revealed that, in all cases, there was a high coverage of residues in the models. All of them showed a very close conformation, indicated by the low root-mean-square deviation (RMSD) values of backbone atoms, which indicate a high similarity among them. However, low affinity between the enzymes and the crocetin dialdehyde were observed. Phylogenetic analysis and binding affinities calculations, including some ALDHs from Gardenia jasmonoides, Crocus sieberi, and Buddleja species that accumulate crocetin and Bixa orellana synthetizing the apocarotenoid bixin selected on their expression pattern matching with the accumulation of either crocins or bixin, pointed out that family 2 C4 members might be involved in the conversion of crocetin dialdehyde to crocetin with high specificity.

Evolutionarily distinct carotenoid cleavage dioxygenases are responsible for crocetin production in Buddleja davidii.

Crocetin, one of the few colored apocarotenoids known in nature, is present in flowers and fruits and has long been used medicinally and as a colorant. Saffron is the main source of crocetin, although a few other plants produce lower amounts of this apocarotenoid. Notably, Buddleja davidii accumulates crocetin in its flowers. Recently, a carotenoid dioxygenase cleavage enzyme, CCD2, has been characterized as responsible for crocetin production in Crocus species. We searched for CCD2 homologues in B. davidii and identified several CCD enzymes from the CCD1 and CCD4 subfamilies. Unexpectedly, two out of the three CCD4 enzymes, namely BdCCD4.1 and BdCCD4.3, showed 7,8;7',8' activity in vitro and in vivo over zeaxanthin. In silico analyses of these enzymes and CCD2 allowed the determination of key residues for this activity. Both BdCCD4 genes are highly expressed during flower development and transcripts levels parallel the accumulation of crocins in the petals. Phylogenetic analysis showed that BdCCD4.2 grouped with almost all the characterized CCD4 enzymes, while BdCCD4.1 and BdCCD4.3 form a new sub-cluster together with CCD4 enzymes from certain Lamiales species. The present study indicates that convergent evolution led to the acquisition of 7,8;7',8' apocarotenoid cleavage activity in two separate CCD enzyme families.

Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis.

Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7',8' double bonds adjacent to a 3-OH-ß-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-ß-apo-8'-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-ß-apo-8'-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the ß-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.

Unraveling Massive Crocins Transport and Accumulation through Proteome and Microscopy Tools during the Development of Saffron Stigma.

Crocins, the glucosides of crocetin, are present at high concentrations in saffron stigmas and accumulate in the vacuole. However, the biogenesis of the saffron chromoplast, the changes during the development of the stigma and the transport of crocins to the vacuole, are processes that remain poorly understood. We studied the process of chromoplast differentiation in saffron throughout stigma development by means of transmission electron microscopy. Our results provided an overview of a massive transport of crocins to the vacuole in the later developmental stages, when electron dense drops of a much greater size than plastoglobules (here defined "crocinoplast") were observed in the chromoplast, connected to the vacuole with a subsequent transfer of these large globules inside the vacuole. A proteome analysis of chromoplasts from saffron stigma allowed the identification of several well-known plastid proteins and new candidates involved in crocetin metabolism. Furthermore, expressions throughout five developmental stages of candidate genes responsible for carotenoid and apocarotenoid biogenesis, crocins transport to the vacuole and starch metabolism were analyzed. Correlation matrices and networks were exploited to identify a series of transcripts highly associated to crocetin (such as 1-Deoxy-d-xylulose 5-phosphate synthase (DXS), 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), carotenoid isomerase (CRTISO), Crocetin glucosyltransferase 2 (UGT2), etc.) and crocin (e.g., ζ-carotene desaturase (ZDS) and plastid-lipid-associated proteins (PLAP2)) accumulation; in addition, candidate aldehyde dehydrogenase (ADH) genes were highlighted.

Tissue-Specific Accumulation of Sulfur Compounds and Saponins in Different Parts of Garlic Cloves from Purple and White Ecotypes.

This study set out to determine the distribution of sulfur compounds and saponin metabolites in different parts of garlic cloves. Three fractions from purple and white garlic ecotypes were obtained: the tunic (SS), internal (IS) and external (ES) parts of the clove. Liquid Chromatography coupled to High Resolution Mass spectrometry (LC-HRMS), together with bioinformatics including Principal Component Analysis (PCA), Hierarchical Clustering (HCL) and correlation network analyses were carried out. Results showed that the distribution of these metabolites in the different parts of garlic bulbs was different for the purple and the white ecotypes, with the main difference being a slightly higher number of sulfur compounds in purple garlic. The SS fraction in purple garlic had a higher content of sulfur metabolites, while the ES in white garlic was more enriched by these compounds. The correlation network indicated that diallyl disulfide was the most relevant metabolite with regards to sulfur compound metabolism in garlic. The total number of saponins was almost 40-fold higher in purple garlic than in the white variety, with ES having the highest content. Interestingly, five saponins including desgalactotigonin-rhamnose, proto-desgalactotigonin, proto-desgalactotigonin-rhamnose, voghieroside D1, sativoside B1-rhamnose and sativoside R1 were exclusive to the purple variety. Data obtained from saponin analyses revealed a very different network between white and purple garlic, thus suggesting a very robust and tight coregulation of saponin metabolism in garlic. Findings in this study point to the possibility of using tunics from purple garlic in the food and medical industries, since it contains many functional compounds which can be exploited as ingredients.

A trial of oral nifedipine and oral labetalol in preeclampsia hypertensive emergency treatment.

This observational retrospective cohort study was conducted to compare oral nifedipine and labetalol for emergency treatment of hypertension in preeclamptic patients. Time (minutes) and necessary doses were outlined to achieve blood pressure lower than 150/95 mmHg. In 14 patients with preeclampsia, 55 hypertensive emergencies were identified (BP >150/95). Of these emergencies, 43 were treated with oral nifedipine 10 mg (10 patients) and 12 with oral labetalol 100 mg (4 patients). To achieve a target blood pressure under 150/95, these doses were repeated as necessary every 20 min, up to a maximum of 4 doses. Oral nifedipine reduced BP more rapidly (31.30 vs. 53.50 min, p = .03). No maternal or foetal adverse events were observed and no major differences were found according to the type of delivery. Oral nifedipine is faster than and at least as safe as labetalol in pre-eclampsia hypertensive emergency treatment.

Intron retention and rhythmic diel pattern regulation of carotenoid cleavage dioxygenase 2 during crocetin biosynthesis in saffron.

The carotenoid cleavage dioxygenase 2, a new member of the CCD family, catalyzes the conversion of zeaxanthin into crocetin-dialdehyde in Crocus. CCD2 is expressed in flowers, being responsible for the yellow, orange and red colorations displayed by tepals and stigma. Three CsCCD2 genes were identified in Crocus sativus, the longest contains ten exons and the shorter is a truncated copy with no introns and which lacks one exon sequence. Analysis of RNA-seq datasets of three developmental stages of saffron stigma allowed the determination of alternative splicing in CsCCD2, being intron retention (IR) the prevalent form of alternative splicing in CsCCD2. Further, high IR was observed in tissues that do not accumulate crocetin. The analysis of one CsCCD2 promoter showed cis-regulatory motifs involved in the response to light, temperature, and circadian regulation. The light and circadian regulation are common elements shared with the previously characterized CsLycB2a promoter, and these shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation of these genes during the development of the stigma in saffron. A daily coordinated rhythmic regulation for CsCCD2 and CsLycB2a was observed, with higher levels of mRNA occurring at low temperatures during darkness, confirming the results obtained in the in silico promoter analysis. In addition, to the light and temperature dependent regulation of CsCCD2 expression, the apocarotenoid ß-cyclocitral up-regulated CsCCD2 expression and could acts as a mediator of chromoplast-to-nucleus signalling, coordinating the expression of CsCCD2 with the developmental state of the chromoplast in the developing stigma.