A transient expression tool box for anthocyanin biosynthesis in Nicotiana benthamiana.

Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3-O-rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside and delphinidin 3-O-glucoside could be obtained in high amounts in a few days. Additionally, co-infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.

Engineering Betalain Biosynthesis in Tomato for High Level Betanin Production in Fruits.

Betalains are pigments found in plants of the Caryophyllales order, and include the red-purple betacyanins and the yellow-orange betaxanthins. The red pigment from red beets, betanin, is made from tyrosine by a biosynthetic pathway that consists of a cytochrome P450, a L-DOPA dioxygenase, and a glucosyltransferase. The entire pathway was recently reconstituted in plants that do not make betalains naturally including potato and tomato plants. The amount of betanin produced in these plants was however not as high as in red beets. It was recently shown that a plastidic arogenate dehydrogenase gene involved in biosynthesis of tyrosine in plants is duplicated in Beta vulgaris and other betalain-producing plants, and that one of the two encoded enzymes, BvADHα, has relaxed feedback inhibition by tyrosine, contributing to the high amount of betanin found in red beets. We have reconstituted the complete betanin biosynthetic pathway in tomato plants with or without a BvADHα gene, and with all genes expressed under control of a fruit-specific promoter. The plants obtained with a construct containing BvADHα produced betanin at a higher level than plants obtained with a construct lacking this gene. These results show that use of BvADHα can be useful for high level production of betalains in heterologous hosts. Unlike red beets that produce both betacyanins and betaxanthins, the transformed tomatoes produced betacyanins only, conferring a bright purple-fuschia color to the tomato juice.

High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns.

The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome. The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs, including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana, we investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLSs), and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one. However, the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence, which dramatically improved the editing efficiency of the constructs. None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes. The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

Does severe acute pain provoke lasting changes in attentional and emotional mechanisms of pain-related processing? A longitudinal study.

Pain experiences, learning, and genetic factors have been proposed to shape attentional and emotional processes related to pain. We aimed at investigating whether a singular major pain experience also changes cognitive-emotional processing. The influence of acute postoperative pain after cosmetic surgery of the thorax was tested in 80 preoperatively pain-free male individuals. Acute pain was measured as independent variable during the first week postsurgery by pain intensity ratings and the requested analgesic boluses (Patient-Controlled Epidural Analgesia (PCEA)). Pain catastrophizing (Pain Catastrophizing Scale (PCS)), pain anxiety (Pain Anxiety and Symptom Scale (PASS)), pain hypervigilance (Pain Vigilance and Awareness Questionnaire (PVAQ)), and attentional biases to emotionally loaded stimuli (including pain) in a dot-probe task were assessed 1 week, 3 months, and 6 months postsurgery as dependent variables. Hierarchical regression analyses were performed to test whether the 2 acute pain parameters can predict these cognitive-emotional variables. As a rigorous test, significant prediction was required in addition to the prediction of the dependent variables by themselves with lag-1. Acute pain (mainly the pain ratings) appeared to be a significant predictor for PCS, PASS, and PVAQ 1 week after surgery (deltaR(2) = [8.7% to 11.3%]). In contrast, the attentional biases in the dot-probe task could not be predicted by the pain ratings. The levels of pain catastrophizing and pain hypervigilance increased in the acute phase after surgery when influenced by acute pain and declined, along with pain anxiety, during the next 3 months. In conclusion, a one-time intense pain experience, such as acute postoperative pain, appeared to produce at least short-lived changes in the attentional and emotional processing of pain.

Does pain necessarily have an affective component? Negative evidence from blink reflex experiments.

BACKGROUND: Experimental pain research has shown that the affective component of pain is influenced strongly by situational characteristics; affective pain processing appears to be particularly pronounced in situations that provoke a feeling of uncertainty and uncontrollability. OBJECTIVES: To determine whether the affective component of pain can be completely abolished if a 'safe', particularly predictable stimulation paradigm is applied. METHODS: Forty healthy volunteers recruited at the University of Bamberg (Bamberg, Germany) were assessed in two experiments. Tonic contact heat stimuli staged in three intensities (warmth, heat and pain) relative to the individual pain threshold was applied; these were predictable with regard to intensity and course, and the subjects had easy access to control. The startle reflex was assessed as an objective measure of affective response. In addition, the subjects provided unpleasantness ratings. To compare these results to a gold standard for affective response, affective pictures taken from the International Affective Picture System were presented during temperature stimulation in the second experiment. RESULTS: Both experiments showed no potentiation of the startle reflex under painful heat stimulation compared with the two nonpainful stimulus intensities (heat and warmth), although the painful stimulation was clearly rated as more unpleasant. CONCLUSIONS: Results suggest that it is possible to develop a 'safe' noxious stimulus, which is rated as clearly unpleasant, but lacks physiological indication of negative affect. This divergence might be explained by subjective ratings being influenced by the instructions. The possibility of reducing the pain affect by suggesting 'safety' may be of therapeutic interest.

Investigating the affective component of pain: no startle modulation by tonic heat pain in startle responsive individuals.

BACKGROUND: Experimental tonic pain has been assumed to equal clinical pain by triggering sizeable affective responses. A psycho-physiological indicator of defensive affective-motivational responses is the startle reflex. However, earlier studies have not provided unequivocal evidence for a potentiation of the startle reflex during tonic contact heat pain. OBJECTIVES: The demonstration of modulating effects of pain on the startle reflex might require very intense tonic stimulation and investigation of subjects, who are particularly sensitive to startle potentiation by threatening cues. METHOD: We investigated a sample of healthy subjects (N=20), who had shown pronounced startle amplitude potentiation in response to attack pictures. Noxious stimulation was provided by hand immersion into a hot water bath, which is a tonic pain model known for intense and summated stimulation. Modulation of the startle reflex was attempted by use of two stimulation intensities (42 °C, 46 °C) and one control condition (no stimulation). RESULTS: Even in these favorable conditions, we did not observe startle potentiation under painful stimulation in comparison to non-painful conditions although subjects reported to be experiencing moderate to high pain. CONCLUSIONS: Our findings indicate that tonic heat pain does not trigger defensive affective-motivational responses as measured by the startle reflex when it is applied in a predictable and thus non-threatening fashion. Future research should investigate the effects of manipulations of threat on startle responses to painful stimulation.

Serum tau protein level as a marker of axonal damage in acute ischemic stroke.

Biochemical markers of brain damage, e.g. ischemic stroke, should reflect the volume of irreversibly damaged brain parenchyma and the clinical outcome in a single patient in order to allow estimation of prognosis at an early stage. Tau protein, which derives predominantly from neurons and axons, is elevated in the cerebrospinal fluid of patients with neurodegenerative disease. This makes tau protein a potential marker of neuronal/axonal injury. In order to test this hypothesis, the current study aimed at showing that tau protein is measurable in the blood after acute ischemic stroke and that it correlates with clinical disability and stroke volume. In a longitudinal prospective study we measured tau protein serum levels with an ELISA in 30 patients longitudinally after ischemic stroke. Tau protein was detectable within 5 days after ischemia in the sera of 7/20 patients with MRI-proven infarction and in 2/10 patients with transitory ischemic attack; both of them had a small infarction visible on the MRI scan. Tau protein was measurable within 6 h after symptom onset, peaked after 3-5 days and correlated with infarct volume and disability after 3 months. In conclusion, serum tau protein is a candidate marker of axonal injury. In stroke, its clinical use is limited, because it is detectable only in a proportion of patients.

Criptococose: tratamento prognóstico e prevençäo / Cryptococosis: treatment, prognostic and prevention

O tratamento da criptococose depende do tecido envolvido, da doença predisponente, do mecanismo de defesa do hospedeiro e dos achados liquóricos e sorológicos. O tratamento de escolha para a meningite criptococócica é a combinaçäo de anfotericina B e 5-flucitosina. O fluconazol, das novas drogas, é a atual indicaçäo para o tratamento de manutençäo, por sua açäo antifúngica e penetraçäo no sistema nervoso central