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Stilbenes are phytoalexins with health-promoting benefits for humans. Here, we boost stilbenes' production, and in particular the resveratrol dehydrodimer viniferin, with significant pharmacological properties, by overexpressing stilbene synthase (STS) under unlimited phenylalanine (Phe) supply. Vitis vinifera cell cultures were co-transformed with a feedback-insensitive E. coli DAHP synthase (AroG*) and STS genes, under constitutive promoters. All transgenic lines had increased levels of Phe and stilbenes (74-fold higher viniferin reaching 0.74 mg/g DW). External Phe feeding of AroG* + STS lines caused a synergistic effect on resveratrol and viniferin accumulation, achieving a 26-fold (1.33 mg/g DW) increase in resveratrol and a 620-fold increase (6.2 mg/g DW) in viniferin, which to date is the highest viniferin accumulation reported in plant cultures. We suggest that this strategy of combining higher Phe availability and STS expression generates grape cell cultures as potential factories for sustainable production of stilbenes with a minor effect on the levels of flavonoids.

Lisianthus (Eustoma grandiflorum), a leading plant in the cut flower industry, is scentless. Here we show that lisianthus flowers have potential to produce several fragrant benzenoid-phenylpropanoids when substrate availability is not limited. To enable hyperaccumulation of substrates for the production of volatile benzenoid-phenylpropanoids, lisianthus commercial hybrid "Excalibur Pink" was transformed via floral dipping with a feedback-insensitive Escherichia coli DAHP synthase (AroG*) and Clarkia breweri benzyl alcohol acetyltransferase (BEAT), under constitutive promoters. The T1 progeny of "Excalibur Pink" plants segregated into four visual phenotypes, with pink or white colored petals and multiple or single petal layers. Interestingly, transformation with AroG* and BEAT caused no significant effect in the pigment composition among phenotypes, but did increase the levels of down-stream fragrant volatile benzenoids. All the transgenic lines exclusively accumulated methyl benzoate, a fragrant benzenoid, either in their petals or leaves. Furthermore, feeding with benzyl alcohol resulted in the accumulation of two novel benzenoids, benzyl acetate (the product of BEAT) and benzoate, as well as a dramatic increase in the concentrations of additional benzenoid-phenylpropanoid volatiles. Presumably, the degree of benzaldehyde overproduction after benzyl alcohol feeding in both leaves and flowers revealed their reverse conversion in lisianthus plants. These findings demonstrate the concealed capability of lisianthus plants to produce a wide array of fragrant benzenoid-phenylpropanoids, given high substrate concentrations, which could in turn open opportunities for future scent engineering.

Flowers are the most vulnerable plant organ to infection by the necrotrophic fungus Botrytis cinerea. Here we show that pre-treatment of chrysanthemum (Chrysanthemum morifolium) flowers with phenylalanine (Phe) significantly reduces their susceptibility to B. cinerea. To comprehend how Phe treatment induces resistance, we monitored the dynamics of metabolites (by GC/LC-MS) and transcriptomes (by RNAseq) in flowers after Phe treatment and B. cinerea infection. Phe treatment resulted in accumulation of 3-phenyllactate and benzaldehyde, and in particular induced the expression of genes related to Ca2+ signaling and receptor kinases, implicating an induction of the defense response. Interestingly, the main effects of Phe treatment were observed in flowers exposed to B. cinerea infection, stabilizing the global fluctuations in the levels of metabolites and transcripts while reducing susceptibility to the fungus. We suggest that Phe-induced resistance is associated to cell priming, enabling rapid and targeted reprogramming of cellular defense responses to resist disease development. After Phe pre-treatment, the levels of the anti-fungal volatiles phenylacetaldehyde and eugenol were maintained and the level of coniferin, a plausible monolignol precursor in cell wall lignification, was strongly increased. In addition, Phe pre-treatment reduced ROS generation, prevented ethylene emission, and caused changes in the expression of a minor number of genes related to cell wall biogenesis, encoding the RLK THESEUS1, or involved in Ca2+ and hormonal signaling processes. Our findings point to Phe pre-treatment as a potential orchestrator of a broad-spectrum defense response which may not only provide an ecologically friendly pest control strategy but also offers a promising way of priming plants to induce defense responses against B. cinerea.

Cold is the best means of prolonging fruit storage. However, tropical fruit are susceptible to cold storage. The mode of action of mango fruit tolerance to suboptimal cold temperature of 7 or 10 °C after postharvest application of 8 mM phenylalanine was investigated using transcriptomic and metabolomic analyses of mango fruit during suboptimal cold storage. Phenylalanine-treated fruit had less chilling injuries-black spot and pitting electrolyte leakage,-and reduced decay after suboptimal cold storage. Phenylalanine treatment induced genes related to plant-pathogen interactions, plant hormone signal transduction, and the phenylpropanoid pathway, increasing the levels of the flavonoids quercetin and kaempferol glycosides and anthocyanins, and antioxidant content. Reduced oxidation led to lower lipid peroxidation, and a reduction in fatty acid-degradation products, e.g., volatile aldehydes. Treatment with phenylalanine, therefore, enhances chilling tolerance of mango fruit through regulation of metabolic and defense-related pathways, maintaining high levels of flavonoids, and antioxidants enzyme activity, and reducing H2O

Stilbenes and flavonoids are two major health-promoting phenylpropanoid groups in grapes. Attempts to promote the accumulation of one group usually resulted in a decrease in the other. This study presents a unique strategy for simultaneously increasing metabolites in both groups in

C. morifolium are obligate short day plants that can be induced to flower throughout the year and their stem length can be controlled by maintaining artificial long days before the onset of flowering. The ability to strictly control the flowering of chrysanthemums enabled this crop to become one of the leading ornamentals grown for the cut flower industry. The most effective inhibition of chrysanthemum flowering was found to be red light, while blue light had no inhibitory effect, and in some studies was even suggested to induce flowering. Here the effect of light quality and duration of day length illumination on chrysanthemum growth and flowering was examined. In contrast to previous studies, the results presented here suggest that blue light does inhibit chrysanthemum flowering, but the efficiency of inhibition is dependent on the duration of the lighting. Overnight illumination with blue LED lights, resulting in inhibition of flowering of three chrysanthemum cultivars, and had no significant effect on their growth. When illumination time was shortened blue light was found to be less efficient than white or red light in preventing flowering. Red LED lights were also tested in a larger scale experiment in a greenhouse, in preventing flowering of two chrysanthemum cultivars. Plants were illuminated for six hours after sundown, with either constant or intermittent red LED lights and harvested two months after the lighting was turned off. Red intermittent light resulted in somewhat shorter stems and lower quality branches in comparison to constant light.

Red-blushed skin color is important for mango fruit quality and marketability. Preharvest spraying of ‘Kent’, 'Shelly' and’ Maya’ mangoes with 0.1, 0.2 or 0.4 % prohydrojasmon (PDJ) induced red skin color (areal coverage and intensity) and anthocyanins accumulation especially in fruit from the outer side of tree canopy, which were exposed to sunlight. Maximum red blush was achieved with 0.2% (‘Shelly’) and 0.4 % (‘Kent’ and ‘Maya’) PDJ in fruit from outside the canopy, with an increase of three fold in anthocyanins and two fold increase in flavonols. HPLC analysis of non-hydrolyzed samples showed that the major anthocyanin peaks were cyanidin-3-galactoside and 7-O-methylcyanidin-3-O-β-D-galactopyranoside, and the major flavonol peaks were glucoside derivatives of quercetin and kaempferol. Thus, PDJ activated the phenylpropanoid and anthocyanin pathways mainly with exposure to sunlight, allowing potential production of specific antioxidant and antifungal flavonols and anthocyanins. Induced red skin color in most of the experiments reduced postharvest decay, which may enhance commercial value and extend shelf life.

Stilbenes are phytoalexins with health-promoting benefits for humans. Here, we boost stilbenes' production, and in particular the resveratrol dehydrodimer viniferin, with significant pharmacological properties, by overexpressing

Mango fruit exposed to sunlight develops red skin and are more resistant to biotic and abiotic stresses. Here we show that harvested red mango fruit that was exposed to sunlight at the orchard is more resistant than green fruit to Colletotrichum gloeosporioides. LCMS analysis showed high amounts of antifungal compounds, as glycosylated flavonols, glycosylated anthocyanins, and mangiferin in red vs. green mango skin, correlated with higher antioxidant and lower ROS. However, also the green side of red mango fruit that has low levels of flavonoids was resistant, indicated induced resistance. Transcriptomes of red and green fruit inoculated on their red and green sides with C. gloeosporioides were analyzed. Overall, in red fruit skin, 2,187 genes were upregulated in response to C. gloeosporioides. On the green side of red mango, upregulation of 22 transcription factors and 33 signaling-related transcripts indicated induced resistance. The RNA-Seq analysis suggests that resistance of the whole red fruit involved upregulation of ethylene, brassinosteroid, and phenylpropanoid pathways. To conclude, red fruit resistance to fungal pathogen was related to both flavonoid toxicity and primed resistance of fruit that was exposed to light at the orchard.

Introduction: The impact of beta blocker use on surgical outcomes of cancer patients receiving immunotherapy is unknown. Hypothesis: Beta blocker use is associated with lower post-operative mortality in cancer patients on immunotherapy. Methods: We used an institutional Advanced Cohort Explorer to identify all patients who underwent any surgical procedure less than 90 days after receiving immunotherapy. Data on the procedure performed as well as the post-operative outcome were collected and analyzed per the pre-operative beta blocker use status. Results: A total of 233 patients underwent surgery between 09/2011-06/2019. Mean age was 64.7 years (range: 16-92). The most common cancer diagnoses were lung (48, 44.4%), melanoma (49, 45.4%) and kidney (11, 10.2%). Immunotherapy medications were pembrolizumab ( 127, 54.5%), nivolumab (51, 21.9%), ipilimumab (43, 18.5%), atezolizumab (11, 4.7%) and avelumab (1, 0.43%). Pre-operative beta blocker use was documented in 140 (60.1%) patients. Most common surgical procedures were skin resection (75, 32.1%), wound repair (47, 20.2%), vascular procedures (36, 15.5%), urethral procedures 16 (6.9%) and sentinel lymph node biopsies (10, 4.3%). The 30-day mortality rate was 10.3% (24 deaths). In a multi-variable logistic regression analysis, pre-operative use of beta blockers was associated with a lower risk of death within 30 days from surgery (OR 0.34, CI 0.13-0.87, P=0.024). 30-day mortality rates were higher in patients undergoing vascular (50%, 17 of 34) versus non-vascular (3.5%, 7 of 199) procedures (P Conclusions: Beta blocker use is associated with a lower 30-day mortality rate after surgical procedures in cancer patients treated with immunotherapy. Further investigation is warranted.

More than 40% of harvested fruit is lost, largely due to decay. In parallel, restrictions on postharvest fungicides call for eco-friendly alternatives. Fruit&rsquo, s natural resistance depends mainly on flavonoids and anthocyanins&mdash, which have antioxidant and antifungal activity&mdash, synthesized from the phenylpropanoid pathway with phenylalanine as a precursor. We hypothesized that phenylalanine could induce fruit&rsquo, s natural defense response and tolerance to fungal pathogens. The postharvest application of phenylalanine to mango and avocado fruit reduced anthracnose and stem-end rot caused by Colletotrichum gloeosporioides and Lasiodiplodia theobromae, respectively. The postharvest application of phenylalanine to citrus fruit reduced green mold caused by Penicillium digitatum. The optimal phenylalanine concentrations for postharvest application were 6 mM for citrus fruits and 8 mM for mangoes and avocadoes. The preharvest application of phenylalanine to strawberries, mangoes, and citrus fruits also reduced postharvest decay. Interestingly, citrus fruit resistance to P. digitatum inoculated immediately after phenylalanine application was not improved, whereas inoculation performed 2 days after phenylalanine treatment induced the defense response. Five hours after the treatment, no phenylalanine residue was detected on/in the fruit, probably due to rapid phenylalanine metabolism. Additionally, in vitro testing showed no inhibitory effect of phenylalanine on conidial germination. Altogether, we characterized a new inducer of the fruit defense response&mdash, phenylalanine. Preharvest or postharvest application to fruit led to the inhibition of fungal pathogen-induced postharvest decay, suggesting that the application of phenylalanine could become an eco-friendly and healthy alternative to fungicides.

The adaptation of the Agrobacterium-mediated floral-dipping technique is limited, to date, to a small number of plants. In this paper, we present the efficient transformation of one of the leading plants in the cut flower industry, lisianthus (Eustoma grandiflorum). This method is approximately 18 months shorter than the known tissue culture-based transformation. The Excalibur Pink cultivar and two additional breeding lines, X-1042 and X-2541, were transformed using three different marker genes (benzyl alcohol acetyltransferase (BEAT) originating from Clarkia breweri, the feedback-insensitive bacterial gene AroG*, and the empty pART27 vector expressing a kanamycin-resistance cassette (nptII)). Genomic transformation was successful in all tested cases with transformation efficiency ranked from 0.2 to 2.9%, which is well in the range of results from Arabidopsis studies. Unlike Arabidopsis, in which floral-dipping transformation was efficient only at a pre-anthesis stage before ovary sealing, lisianthus flowers were transformed when dipping occurred 4 days pre-anthesis or 3-5 days post-anthesis with 1.5 and 3.7% efficiencies, respectively. Post-anthesis transformation occurred when the flower ovaries were sealed. Flower dipping of Excalibur Pink flowers with fluorescent Agrobacterium containing a GFP marker gene demonstrated Agrobacterium entrance into the sealed flower ovary through the open stigma and style tube. In this study, we demonstrated floral-dipping transformation of a commercial plant, lisianthus Excalibur Pink, occurring after sealing of the ovaries, probably via the stigma and wide open style tunnel.

Fragrance is a desirable characteristic for cut flowers, but is rare among commercial cultivars. To date, there are no transformed commercial flower cultivars with increased fragrance, despite the fact that genetic engineering can generate fragrance in ornamentals without compromising on other commercial traits. One of the major volatile groups responsible for fragrance of flowers such as roses, petunia, snapdragon and clarkia, are the phenylalanine-derived benzenoid phenylpropanoids (BPVs). Here we show that a variety of commercial flowers, belonging to taxonomically distant plant species have the potential of producing fragrant BPVs when treated with exogenous phenylalanine. This group of flowers includes chrysanthemums, roses, anemones, Ornithogalum dubium and gerberas. Chrysanthemums, among the five leading flowers in the cut flower industry, lack flowery fragrance. However, treatment of cut chrysanthemums with phenylalanine resulted in an increase in BPVs preexisting in the flowers, producing a flowery fragrance clearly distinguished by a sensory panel. Similarly, phenylalanine treatment of anemones, also lacking fragrance, resulted in fragrant flowers. However, unlike chrysanthemums, in anemones, in addition to increasing preexisting BPVs, phenylalanine treatment resulted in production of new BPVs, not detected in non-treated flowers. Production of novel compounds due to phenylalanine treatment suggests that concealed metabolic pathways do exist in plants, may be activated by increased substrate availability. This study presents the potential of phenylalanine treatment as a way for increasing flowery fragrance in a large variety of non-fragrant commercially important plants.

Fruit defense against pathogens relies on induced and preformed mechanisms. The present contribution evaluated performed resistance of red and green mango fruit against the fungal pathogen Colletotrichum gloeosporioides and identified the main active antifungal components. High-performance liquid chromatography analysis of nonhydrolyzed mango peel extracts identified major anthocyanin peaks of glycosylated cyanidin and methylcyanidin, and flavonol peaks of glycosylated quercetin and kaempferol, which were more abundant on the 'red side' of red mango fruit. Organic extracts of red vs green mango peel were more efficient in inhibiting C. gloeosporioides. Transcriptome analysis of the mango-C. gloeosporioides interaction showed increased expression of glucosidase genes related to both fungal pathogenicity and host defense. Glucosidase treatment of organic peel extract increased its antifungal activity. Additionally, quercetin and cyanidin had significantly higher antifungal activity than their glycosylated derivatives. Peel extract volatiles treated with glucosidase had antifungal activity. GCMS analysis identified 15 volatiles after glucosidase hydrolysis, seven of them present only in red fruit. These results suggest that the fruit obtains a concealed arsenal of glycosylated flavonoids in its peel when they are hydrolyzed by β-glucosidase that is induced in both fungus and host during infection process, become more toxic to the fungal pathogen, inhibiting decay development.

Botrytis cinerea is a major plant pathogen, causing losses in crops during growth and storage. Here we show that increased accumulation of phenylalanine (Phe) and Phe-derived metabolites in plant leaves significantly reduces their susceptibility to B. cinerea. Arabidopsis, petunia and tomato plants were enriched with Phe by either overexpressing a feedback-insensitive E.coli DAHP synthase (AroG*), or by spraying or drenching detached leaves or whole plants with external Phe, prior to infection with B. cinerea. Metabolic analysis of Arabidopsis and petunia plants overexpressing AroG* as well as wt petunia plants treated externally with Phe, revealed an increase in Phe-derived phenylpropanoids accumulated in their leaves, and specifically in those inhibiting B. cinerea germination and growth, suggesting that different compounds reduce susceptibility to B. cinerea in different plants. Phe itself had no inhibitory effect on germination or growth of B. cinerea, and inhibition of Phe metabolism in petunia plants treated with external Phe prevented decreased susceptibility to the fungus. Thus, Phe metabolism into an array of metabolites, unique to each plant and plant organ, is the most probable cause for increased resistance to Botrytis. This mechanism may provide a basis for ecologically friendly control of a wide range of plant pathogens.

Pediatric brain tumors with replication repair deficiency (RRD) are hypermutant and may respond favorably to immune checkpoint inhibition (ICI). We are collecting ongoing clinical and molecular data from patients with RRD hypermutant cancers treated with ICI as a part of our consortium registry study. Companion biomarkers include tumor mutational burden (TMB), neoantigens and genetic signatures obtained from whole genome and exome sequencing. Immune inference is obtained from RNAseq. Additionally, T-cell receptor rearrangement data are collected from the tumor and blood throughout treatment. From 2015–2018, 53 patients were treated with ICI and combination therapies. Of these 39 had brain tumors, with 93% having high grade gliomas. Two-year overall survival for the entire cohort is 47+/-8%, which compares favorably with historical controls. Tumor location had major impact on outcome. Non-CNS solid tumor patients have 2-year OS of 78+/-11%, all failures occurring within the first 2 months, and sustained responses are observed for 3 years. In contrast, OS for brain tumors is 39+/-10% with late recurrences observed even after 2 years of therapy (p=0.02). Large tumor size and total tumor burden are associated with higher rates of “flare” and poor outcome throughout all cancers. While all tumors are hypermutant, TMB and predicted neoantigens do not correlate with response. However, specific signatures extracted from SNVs and indels are significantly associated with response to ICI and favorable outcome (p=0.005). Notably, RRD IDH1 mutant glioblastomas have particularly poor response to ICI. Interestingly, glioblastomas (n=8) which failed single agent ICI had favorable responses to combination immunotherapies with prolonged survival of 65%+/-8% one year after failure vs 0 for untreated patients (p=0.01). The favorable outcome and responses to ICI are encouraging. Since brain tumors respond less favorably to ICI than other solid tumors, combination therapies based on tumor and immune signatures of these cancers may be beneficial.

Position sensors, such as the gyroscope, the magnetometer and the accelerometer, are found in a staggering variety of devices, from smartphones and UAVs to autonomous robots. Several works have shown how adversaries can mount spoofing attacks to remotely corrupt or even completely control the outputs of these sensors. With more and more critical applications relying on sensor readings to make important decisions, defending sensors from these attacks is of prime importance. In this work we present practical software based defenses against attacks on two common types of position sensors, specifically the gyroscope and the magnetometer. We first characterize the sensitivity of these sensors to acoustic and magnetic adversaries. Next, we present two software-only defenses: a machine learning-based single sensor defense, and a sensor fusion defense which makes use of the mathematical relationship between the two sensors. We performed a detailed theoretical analysis of our defenses, and implemented them on a variety of smartphones, as well as on a resource-constrained IoT sensor node. Our defenses do not require any hardware or OS-level modifications, making it possible to use them with existing hardware. Moreover, they provide a high detection accuracy, a short detection time and a reasonable power consumption.

Pediatric brain tumors with replication repair deficiency (RRD) are hypermutant and may respond to immune checkpoint inhibition (ICI). We performed a consortium registry study of ICI in recurrent RRD cancers. Clinical and companion biomarkers were collected longitudinally on all patients. Biomarkers included tumor mutational burden (TMB), neoantigens and genetic signatures obtained from whole genome and exome sequencing. Immune inference was obtained by RNAseq and T cell rearrangement was collected in the tumor and in blood throughout treatment. Of the 46 tumors on the study, 32 were brain tumors with glioblastoma in 96%. Rapid, objective responses (>50%) were observed in 50% of glioblastomas. Three year overall survival for the whole cohort was 48+/-8% which compares favorably with historical controls. Brain tumors fared worse with OS of 39+/-10% and late recurrences observed even after 2 years of therapy (p=0.02). Tumor size and acute “flare” constitute poor outcome throughout all cancers. While all tumors are hypermutant, TMB and predicted neoantigens correlated with response to ICI (p=0.02). Specific signatures extracted from SNVs and total mutations predicted response to ICI and favorable outcome (p=0.005). RNA inference and TCR reveal that the FLARE phenotype is mostly acute nonspecific immune response and not true progression. Finally, glioblastomas (n=8) which failed single agent ICI had favorable responses to combinational immunotherapies with prolonged survival of 65%+/-8% at one year after failure vs 0 for other patients (p=0.01). RRD glioblastomas exhibit favorable outcome and responses to ICI. Combinational therapies based on tumor and immune signatures of these cancers are necessary.

Growth in hot climates selectively alters potato tuber secondary metabolism—such as the anthocyanins, carotenoids, and glycoalkaloids—changing its nutritive value and the composition of health-promoting components. Potato breeding for improved nutritional value focuses mainly on increasing the health-promoting carotenoids and anthocyanins, and controlling toxic steroidal glycoalkaloids (SGAs). Metabolite levels are genetically determined, but developmental, tissue-specific, and environmental cues affect their final content. Transcriptomic and metabolomic approaches were applied to monitor carotenoid, anthocyanin, and SGA metabolite levels and their biosynthetic genes’ expression under heat stress. The studied cultivars differed in tuber flesh carotenoid concentration and peel anthocyanin concentration. Gene expression studies showed heat-induced downregulation of specific genes for SGA, anthocyanin, and carotenoid biosynthesis. KEGG database mapping of the heat transcriptome indicated reduced gene expression for specific metabolic pathways rather than a global heat response. Targeted metabolomics indicated reduced SGA concentration, but anthocyanin pigments concentration remained unchanged, probably due to their stabilization in the vacuole. Total carotenoid level did not change significantly in potato tuber flesh, but their composition did. Results suggest that growth in hot climates selectively alters tuber secondary metabolism, changing its nutritive value and composition of health-promoting components.

Plants produce a diverse repertoire of specialized metabolites that have multiple roles throughout their life cycle. Some of these metabolites are essential components of the aroma and flavor of flowers and fruits. Unfortunately, attempts to increase the yield and prolong the shelf life of crops have generally been associated with reduced levels of volatile specialized metabolites and hence with decreased aroma and flavor. Thus, there is a need for the development of new varieties that will retain their desired traits while gaining enhanced scent and flavor. Metabolic engineering holds great promise as a tool for improving the profile of emitted volatiles of domesticated crops. This mini review discusses recent attempts to utilize metabolic engineering of the phenylpropanoid and its primary precursor pathway to enhance the aroma and flavor of flowers and fruits.

This study aims to compare the clinical and procedural characteristics, including iatrogenic complications, of transradial versus transfemoral approach in patients who underwent peripheral arterial interventions. We retrospectively analyzed data of 72 patients who had undergone interventions of peripheral arteries in the preceding 3 years. Of all the procedures, 39 were performed using the transfemoral approach and 33 using the transradial approach. We assessed baseline clinical factors as well as procedure-related parameters (volume of contrast media, the amount of radiation, and duration of radiation exposure), rates of primary success, and acute vascular, access-site, or neurological complications. Patients whose interventions were done via transradial access had similar demographic characteristics to those who had transfemoral angioplasties. The presence of cardiovascular risk factors was similar in the two groups, but patients in the transradial subset were significantly more likely to have dyslipidemia (90.9 vs.71.7%, p value = 0.04) and less likely to have baseline chronic kidney disease (3.0 vs. 23.0%, p value = 0.01). The length of admission was shorter for patients who underwent transradial interventions compared with the transfemoral approach (1.1 vs. 1.3 days, p value = 0.04). Fluoroscopy time, the amount of radiation, and the volume of contrast media were similar between the two groups. Rates of primary success and periprocedural complications did not differ between the two groups. The transradial route appears to be a viable, safe, and noninferior alternative to the transfemoral approach for treatment of atherosclerotic stenosis in peripheral arterial territories. Prospective studies are needed.

Phenylalanine is a precursor for a large group of plant specialized metabolites, including the fragrant volatile benzenoid-phenylpropanoids. In plants, the main pathway leading to production of phenylalanine is via arogenate, while the pathway via phenylpyruvate is considered merely an alternative route. Unlike plants, in most microorganisms the only pathway leading to the synthesis of phenylalanine is via phenylpyruvate. Here we studied the effect of increased phenylpyruvate production in petunia on the formation of benzenoid-phenylpropanoids volatiles and other specialized metabolites originating from phenylalanine both in flowers and leaves. Stimulation of the pathway via phenylpyruvate was achieved by transforming petunia with PheA*, a gene encoding a bacterial feedback insensitive bi-functional chorismate mutase/prephenate dehydratase enzyme. PheA* overexpression caused dramatic increase in the levels of flower benzenoid-phenylpropanoid volatiles such as phenylacetaldehyde, benzaldehyde, benzylacetate, vanillin and eugenol. All three benzenoid-phenylpropanoid pathways characterized in petunia flowers were stimulated in PheA* flowers. In contrast, PheA* overexpression had only a minor effect on the levels of amino acids and nonvolatile metabolites both in the leaves and flowers. The one exception is a dramatic increase in the level of rosmarinate, a conjugate between Phe-derived caffeate and Tyr-derived 3,4-dihydroxyphelylactate, in PheA* leaves. PheA* petunia flowers may serve as an excellent system for revealing the role of phenylpruvate in the production of benzenoid-phenylpropanoids, including possible routes directly converting phenylpyruvate to the fragrant volatiles. This study emphasizes the potential of the phenylyruvate route in achieving fragrance enhancement in flowering plants.

Summary In contrast to detailed knowledge regarding the biosynthesis of anthocyanins, the largest group of plant pigments, little is known about their in planta degradation. It has been suggested that anthocyanin degradation is enzymatically controlled and induced when beneficial to the plant. Here we investigated the enzymatic process in Brunfelsia calycina flowers, as they changed color from purple to white. We characterized the enzymatic process by which B. calycina protein extracts degrade anthocyanins. A candidate peroxidase was partially purified and characterized and its intracellular localization was determined. The transcript sequence of this peroxidase was fully identified. A basic peroxidase, BcPrx01, is responsible for the in planta degradation of anthocyanins in B. calycina flowers. BcPrx01 has the ability to degrade complex anthocyanins, it co-localizes with these pigments in the vacuoles of petals, and both the mRNA and protein levels of BcPrx01 are greatly induced parallel to the degradation of anthocyanins. Both isoelectric focusing (IEF) gel analysis and 3D structure prediction indicated that BcPrx01 is cationic. Identification of BcPrx01 is a significant breakthrough both in the understanding of anthocyanin catabolism in plants and in the field of peroxidases, where such a consistent relationship between expression levels, in planta subcellular localization and activity has seldom been demonstrated.

SummaryReducing light levels, decreasing the red to far-red (R:FR) light ratio, and exposing stock plants to blue light have been shown to increase the rooting potential of cuttings in various species. Here, we tested the effects of growing stock plants under photoselective shade nets, altering the spectrum of transmitted sunlight, and/or enriching the relative level of scattered light, on the yield and rooting quality of cuttings. Three ornamental plants, Pelargonium (including three cultivars, ‘Isabel’, ‘Alara’ and ‘De Capo’), Laurus nobilis, and Heteromelis normally propagated by rooting cuttings, were chosen for this study. The nets chosen were a black neutral net and pearl, blue, or yellow photoselective nets. In Heteromelis, the pearl net caused a significant increase in the number of rooted cuttings per plant, but had no effect on the number of rooted cuttings in Pelargonium or L. nobilis stock plants. Instead, significant increases in cutting yields in all three Pelargonium cultivars and in L. nob...

Patients with constitutional mismatch repair deficiency (CMMRD) are prone to developing high-grade glioma (HGG). These tumours acquire DNA polymerase mutations and become ultra-hypermutant harbouring hundreds of mutations per megabase. The impact of these mutations on methylation profile and the ability of the tool to differentiate MMRD tumours from others is unknown. In order to answer these questions, we performed either 450k/850K methylation analysis on a cohort of 52 CMMRD-HGG and compared them to 148 non-CMMRD HGG and normal brain controls. CMMRD HGG harbouring classic mutations in histone 3 or IDH genes had a methylation profile which clustered closely with non-MMRD tumours harbouring these mutations. Tumours without these alterations exhibited a tendency to hypomethylation with some tumours being extremely hypomethylated in comparison to other HGG. Hypomethylation was unrelated to mutational burden and type of DNA polymerase mutation present. Gene set analysis of methylation patterns revealed enrichment of hypomethylation for cellular pathways involved in cellular metabolism, organelle maintenance, mitotic cell cycle and gene expression. This pattern persisted in subgroup analysis of IDH mutant tumours in patients with and without MMRD. Importantly, this pattern was present in MMRD HGG with mutational burdens

PCSK9-i are gaining traction as effective lipid-lowering agents. We hypothesized that female patients were under-represented in landmark clinical trials of PCSK9-i. In this meta-analysis, we searched and reviewed all currently available randomized clinical trials (RCTs) of PCSK9-i. We defined the

Identifying the cardiotoxic potential of new anti-cancer drugs is essential in order to balance efficacy and toxicity. We performed Pubmed search of 4 chemotherapy drugs and ‘cardiotoxicity’. Of these 4 drugs, 2 have a known association with cardiac toxicity. We created plots representing the

Dodonaea ‘Dana’ is a natural hybrid (Dodonaea viscosa purpurea x Dodonaea aptera) cultivated for the production of decorative cut branches bearing dark red foliage with red fruit. This hybrid flowers throughout the year in the Mediterranean climate, but fruit set occurs only during the winter, leading to a short two month period of fruit bearing branches. The aim of this work was to characterize the environmental conditions affecting fruit set in D. ‘Dana’ plants, and improve the efficiency under sub-optimal conditions by hormonal treatment of the plants. When plants were grown at different temperature regimes the number of fruit developed from each inflorescence was 3-fold higher in plants grown at 17 °C/9 °C, in comparison to 29 °C/21 °C, suggesting that temperature is a critical factor for the development of D. ‘Dana’ fruit. In addition, the anthocyanin concentration of fruit grown at the lower temperature regime was four times higher than those grown at the elevated temperature. To improve fruit setting in plants grown at moderate or elevated temperatures (23 °C/15 °C and 29 °C/21 °C), individual inflorescences were treated with one of the two synthetic auxins, naphthalene-1-acetic acid (NAA) and 4-chlorophenoxy acetic acid (4-CPA). Both auxins increased the number of set fruit per inflorescence at both temperature regimes, with an advantage to the NAA treatment at the 23 °C/15 °C regime. Treatment of whole plants with either NAA or 4-CPA at 26 °C/18 °C (day/night respectively) regime, caused a significant increase (2.5–3-fold) in the number of inflorescences per branch with no visual effect on the vegetative characteristics of the plants. In addition, NAA treatment caused an increase of over 50% in the number of flowers per inflorescence. Similar to the treatment of individual inflorescences, here too both synthetic auxins improved the yield of fruit setting.

Plant vacuolar peroxidases catalyze the reduction of toxic H(2)O(2) accumulated in the vacuoles by oxidizing a variety of secondary metabolites. The redundancy of peroxidases and their ability to react with a wide range of substrates have prevented the observation of a clear phenotypic effect by modifying a single gene. Here we review the correlative and partial data on vacuolar peroxidases, including evidence for genes encoding vacuolar localized peroxidases, and indications of peroxidase activity in the vacuole. Based on these data, we suggest that these enzymes are key players in the adaptation of plants to change and serve as plant caretakers. At the cellular level, peroxidases protect the plant by scavenging excess H(2)O(2) that accumulates in the vacuoles under stressful conditions. At the tissue level, they are responsible for the last steps in the synthesis of the phytoalexins that often accumulate following pathogen attack of the plant tissue. At the whole-plant level, we suggest that peroxidases are involved in controlling the quality and quantity of light reaching the photosynthetic apparatus as plants adapt to lower light intensities. Further characterization of peroxidases, based on high-throughput genomic and metabolomic data, will help elucidate the mechanisms by which plants adapt to change.

Summary‘Crimson Seedless’ is a red, seedless table grape (Vitis vinifera) variety which may not develop adequate colour during hot weather. This problem can be alleviated, in part, by applying abscisic acid (ABA). We were interested to determine whether a gibberellin (GA3), which is used to improve the size of grape berries, might interfere with this response. We were also interested in examining whether short periods of high temperature would affect the response to ABA. In addition, we assessed the optimum time at which to apply ABA, and the role of ethylene in berry colour development. These experiments were conducted in commercial vineyards in Israel, and on detached berries held under controlled conditions. GA3 had no effect on the response to ABA. There was a mixed response to ethylene, and to the application of ethylene inhibitors in the vineyard or in controlled studies in growth chambers. The best response in terms of colour development (i.e., anthocyanin levels) on detached berries occurred when ...

Anthocyanins are the largest and best studied group of plant pigments. However, not very much is known about the fate of these phenolic pigments after they have accumulated in the cell vacuoles of plant tissues. We have previously shown that magnesium treatment of ornamentals during the synthesis of anthocyanins in the flowers or foliage caused an increase in the pigment concentration. In this study, we characterized the effect of magnesium on the accumulation of anthocyanin in red cell suspension originating from Vitis vinifera cv. Gamay Red grapes. Magnesium treatment of the cells caused a 2.5- to 4.5-fold increase in anthocyanin concentration, with no substantial induction of the biosynthetic genes. This treatment inhibited the degradation of anthocyanins occurring in the cells, and changed the ratio between different anthocyanins determining cell color, with an increase in the relative concentration of the less stable pigment molecules. The process by which magnesium treatment affects anthocyanin accumulation is still not clear. However, the results presented suggest at least part of its effect on anthocyanin accumulation stems from inhibition of the pigments' catabolism. When anthocyanin biosynthesis was inhibited, magnesium treatments prevented the constant degradation of anthocyanins in the cell suspension. Future understanding of the catabolic processes undergone by anthocyanins in plants may enable more efficient inhibition of this process and increased accumulation of these pigments, and possibly of additional phenolic compounds.

PCSK9 inhibitors have recently been shown to improve the incidence of numerous cardiovascular outcomes. As further investigations are under way, these novel drugs are destined to augment the current battery of lipid-lowering medications. Nonetheless, the cost of PCSK9 inhibitors is alarmingly high

Dry legumes are staple and potentially functional food, being a good source of polyphenols, flavonoids, and antioxidant activity. The objective of this study was to determine the total polyphenol content (TPC), total flavonoid content (TFC), and their relation with antioxidant capacity in 17 chickpea lines having colored seed coats (black, red, brown, green, rubiginous, gray, yellow, cream, or beige). The seed coat usually contains more than 95% of these compounds. In this study, both TPC and TFC varied significantly among different lines and were highly correlated to antioxidant activity. Colored seeds contained up to 13-, 11-, and 31-fold more TPC, TFC, and antioxidant activity, respectively, than cream- and beige-color seeds. Thus, colored chickpea could be a potentially functional food in addition to its traditional role of providing dietary proteins and dietary fibers.

Brunfelsia calycina flowers change colour from purple to white due to anthocyanin degradation, parallel to an increase in fragrance and petal size. Here it was tested whether the production of the fragrant benzenoids is dependent on induction of the shikimate pathway, or if they are formed from the anthocyanin degradation products. An extensive characterization of the events taking place in Brunfelsia flowers is presented. Anthocyanin characterization was performed using ultraperfomance liquid chromatography-quadrupole time of flight-tandem mass specrometry (UPLC-QTOF-MS/MS). Volatiles emitted were identified by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Accumulated proteins were identified by 2D gel electrophoresis. Transcription profiles were characterized by cross-species hybridization of Brunfelsia cDNAs to potato cDNA microarrays. Identification of accumulated metabolites was performed by UPLC-QTOF-MS non-targeted metabolite analysis. The results include characterization of the nine main anthocyanins in Brunfelsia flowers. In addition, 146 up-regulated genes, 19 volatiles, seven proteins, and 17 metabolites that increased during anthocyanin degradation were identified. A multilevel analysis suggests induction of the shikimate pathway. This pathway is the most probable source of the phenolic acids, which in turn are precursors of both the benzenoid and lignin production pathways. The knowledge obtained is valuable for future studies on degradation of anthocyanins, formation of volatiles, and the network of secondary metabolism in Brunfelsia and related species.

A protocol for plantlet regeneration through shoot formation was developed for the neotropical shrub Brunfelsia calycina. This shrub is unique in its change in flower color from dark purple to white. Explants from young and mature leaves were incubated on MS medium (pH 5.7, 30 g/l sucrose, 7.5 g/l agar) with various combinations of Indole-3-acetic acid (IAA) and 6-Benzyladenine (BA) under a 16 h photoperiod at a constant temperature of 25°C. Shoot emergence was best at 4.44 μM BA and 2.85 μM IAA for young leaf explants, and at 8.88 μM BA, 2.85 μM IAA for mature leaf explants. When shoots were transferred to MS medium supplemented with 1.23–2.46 μM indole butrytic acid (IBA), they developed roots.

In contrast to the detailed knowledge available on anthocyanin synthesis, very little is known about its stability and catabolism in plants. Here we review evidence supporting in planta turnover and degradation of anthocyanins. Transient anthocyanin accumulation and disappearance during plant development or changes in environmental conditions suggest that anthocyanin degradation is controlled and induced when beneficial to the plant. Several enzymes have been isolated that degrade anthocyanins in postharvest fruit that may be candidates for in vivo degradation. Three enzyme groups that control degradation rates of anthocyanins in fruit extracts and juices are polyphenol oxidases, peroxidases and β-glucosidases. Evidence supporting the involvement of peroxidases and β-glucosidases in in vivo anthocyanin degradation in Brunfelsia flowers is presented. Understanding the in vivo anthocyanin degradation process has potential for enabling increased pigmentation and prevention of color degradation in crops.

The tomato INTENSE PIGMENT (IP) genotype is characterized by intense visual pigmentation of unripe and ripe fruits, not thoroughly analyzed thus far. This study was therefore designed to analyze key morphologic, metabolomic, and photomorphogenic phenotypes of this genotype in comparison to its near-isogenic normal counterpart and to evaluate its significance relative to other tomato mutants known for increased fruit pigmentation. The IP genotype produced smaller and darker red fruits, and a substantially increased chloroplast biogenesis was found in its green fruit and leaf tissues. Ripe-red fruits of the IP genotype produced 34-38% more soluble solids and up to 62.6% more carotenoids, but no differences were found in the concentration of flavonoid compounds in its peel tissue. The IP genotype was found to display a greater hypocotyl inhibition response to blue and yellow light, but a more prominent and novel response to total darkness. As a whole, the IP genotype exhibited highly desirable traits, making it a valuable genotype for tomato breeders attempting to introduce functional and taste qualities into tomato fruits.

Summary‘Crimson Seedless’ is a high quality, red, table grape (Vitis vinifera) cultivar, which may fail to develop adequate red colour in warm climates. In addition, most bunches contain some green berries when the rest of the bunch has become red. Abscisic acid (ABA) is a plant hormone which increases in grape berry skin at the onset of maturation and is involved in the regulation of anthocyanin accumulation. A commercial formulation, S-ABA (ProTone®), was sprayed at 400 mg l−1 or 800 mg l−1 in a vineyard at the beginning of veraison, or 200 mg l−1, 400 mg l−1, or 600 mg l−1 S-ABA was applied to detached berries or to small bunches (five-to-six berries) in the laboratory by spraying or by allowing uptake through the pedicel. In the vineyard, the application of S-ABA affected berry colour, changing the red berry colour of the control [hue angle (h°) = 3.6] to black for S-ABA-treated clusters (h° = –45°). Ripeness parameters (soluble solids content, titratable acidity, and berry size) were not affected by ...