Evaluation of genome and base editing tools in maize protoplasts.

Introduction: Despite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing remains a labor-intensive process requiring often several months of in vitro culture to obtain mutant plantlets. To avoid a waste in time and money and to test, in only a few days, the efficiency of molecular constructs or novel Cas9 variants (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9) prior to stable transformation, rapid analysis tools are helpful. Methods: To this end, a streamlined maize protoplast system for transient expression of CRISPR/Cas9 tools coupled to NGS (next generation sequencing) analysis and a novel bioinformatics pipeline was established. Results and discussion: Mutation types found with high frequency in maize leaf protoplasts had a trend to be the ones observed after stable transformation of immature maize embryos. The protoplast system also allowed to conclude that modifications of the sgRNA (single guide RNA) scaffold leave little room for improvement, that relaxed PAM (protospacer adjacent motif) sites increase the choice of target sites for genome editing, albeit with decreased frequency, and that efficient base editing in maize could be achieved for certain but not all target sites. Phenotypic analysis of base edited mutant maize plants demonstrated that the introduction of a stop codon but not the mutation of a serine predicted to be phosphorylated in the bHLH (basic helix loop helix) transcription factor ZmICEa (INDUCER OF CBF EXPRESSIONa) caused abnormal stomata, pale leaves and eventual plant death two months after sowing.

Enhanced pain facilitation rather than impaired pain inhibition in burning mouth syndrome female patients.

BACKGROUND: Deficient endogenous pain modulation has been implicated in the development and exacerbation of chronic orofacial pain. To date, relatively little is known regarding the function of the endogenous pain modulation in patients with burning mouth syndrome (BMS). This case-control study investigated endogenous pain modulation in women with BMS. METHODS: Conditioned pain modulation (CPM) was assessed upon temporal summation (TSP) of thermal pain. Forty female subjects, 20 BMS patients and 20 age-matched control subjects, were included in a 2 session-protocol. Mechanical and thermal pain thresholds were measured on the forearm and hand. TSP was obtained using repetitive laser-evoked thermal stimuli applied on the non-dominant hand, at an intensity yielding to moderate pain. During TSP, CPM was produced by immersing the contralateral foot in a water bath at painful cold (8 °C) temperature. In control conditions, the foot was immersed in a water bath at not painful (30 °C) temperature. RESULTS: BMS was not associated with any impairment in thermal as well as mechanical extracephalic pain thresholds. TSP and CPM efficacy were similar in BMS patients and control subjects. However, BMS patients exhibited enhanced extracephalic heat hyperalgesia. CONCLUSION: This study reveals that there is no impairment of endogenous pain inhibition mechanisms in BMS patients, but rather an increase in pain facilitation.

Consequences of Mixotrophy on Cell Energetic Metabolism in Microchloropsis gaditana Revealed by Genetic Engineering and Metabolic Approaches.

Algae belonging to the Microchloropsis genus are promising organisms for biotech purposes, being able to accumulate large amounts of lipid reserves. These organisms adapt to different trophic conditions, thriving in strict photoautotrophic conditions, as well as in the concomitant presence of light plus reduced external carbon as energy sources (mixotrophy). In this work, we investigated the mixotrophic responses of Microchloropsis gaditana (formerly Nannochloropsis gaditana). Using the Biolog growth test, in which cells are loaded into multiwell plates coated with different organic compounds, we could not find a suitable substrate for Microchloropsis mixotrophy. By contrast, addition of the Lysogeny broth (LB) to the inorganic growth medium had a benefit on growth, enhancing respiratory activity at the expense of photosynthetic performances. To further dissect the role of respiration in Microchloropsis mixotrophy, we focused on the mitochondrial alternative oxidase (AOX), a protein involved in energy management in other algae prospering in mixotrophy. Knocking-out the AOX1 gene by transcription activator-like effector nuclease (TALE-N) led to the loss of capacity to implement growth upon addition of LB supporting the hypothesis that the effect of this medium was related to a provision of reduced carbon. We conclude that mixotrophic growth in Microchloropsis is dominated by respiratory rather than by photosynthetic energetic metabolism and discuss the possible reasons for this behavior in relationship with fatty acid breakdown via ß-oxidation in this oleaginous alga.

Transcriptional frameshifts contribute to protein allergenicity.

Transcription infidelity (TI) is a mechanism that increases RNA and protein diversity. We found that single-base omissions (i.e., gaps) occurred at significantly higher rates in the RNA of highly allergenic legumes. Transcripts from peanut, soybean, sesame, and mite allergens contained a higher density of gaps than those of nonallergens. Allergen transcripts translate into proteins with a cationic carboxy terminus depleted in hydrophobic residues. In mice, recombinant TI variants of the peanut allergen Ara h 2, but not the canonical allergen itself, induced, without adjuvant, the production of anaphylactogenic specific IgE (sIgE), binding to linear epitopes on both canonical and TI segments of the TI variants. The removal of cationic proteins from bovine lactoserum markedly reduced its capacity to induce sIgE. In peanut-allergic children, the sIgE reactivity was directed toward both canonical and TI segments of Ara h 2 variants. We discovered 2 peanut allergens, which we believe to be previously unreported, because of their RNA-DNA divergence gap patterns and TI peptide amino acid composition. Finally, we showed that the sIgE of children with IgE-negative milk allergy targeted cationic proteins in lactoserum. We propose that it is not the canonical allergens, but their TI variants, that initiate sIgE isotype switching, while both canonical and TI variants elicit clinical allergic reactions.

Single and multiple gene knockouts by CRISPR-Cas9 in maize.

KEY MESSAGE: The analysis of 93 mutant alleles in 18 genes demonstrated that CRISPR-Cas9 is a robust tool for targeted mutagenesis in maize, permitting efficient generation of single and multiple knockouts. CRISPR-Cas9 technology is a simple and efficient tool for targeted mutagenesis of the genome. It has been implemented in many plant species, including crops such as maize. Here we report single- and multiple-gene mutagenesis via stably transformed maize plants. Two different CRISPR-Cas9 vectors were used allowing the expression of multiple guide RNAs and different strategies to knockout either independent or paralogous genes. A total of 12 plasmids, representing 28 different single guide RNAs (sgRNAs), were generated to target 20 genes. For 18 of these genes, at least one mutant allele was obtained, while two genes were recalcitrant to sequence editing. 19% (16/83) of mutant plants showed biallelic mutations. Small insertions or deletions of less than ten nucleotides were most frequently observed, regardless of whether the gene was targeted by one or more sgRNAs. Deletions of defined regions located between the target sites of two guide RNAs were also reported although the exact deletion size was variable. Double and triple mutants were created in a single step, which is especially valuable for functional analysis of genes with strong genetic linkage. Off-target effects were theoretically limited due to rigorous sgRNA design and random experimental checks at three potential off-target sites did not reveal any editing. Sanger chromatograms allowed to unambiguously class the primary transformants; the majority (85%) were fully edited plants transmitting systematically all detected mutations to the next generation, generally following Mendelian segregation.

A pilot study of total and allergen-specific IgE serum levels during anestrous, estrous and pregnancy in healthy female dogs.

BACKGROUND: Allergen-specific IgE serology is used for the determination of sensitization status in dogs with atopic dermatitis; the influence of the female reproductive cycle on the results of such methods has not been studied in dogs. OBJECTIVES: To compare the total and allergen-specific IgE of healthy bitches during anestrous, estrous and pregnancy. ANIMALS: Eight privately owned, healthy bitches. METHODS: Total and allergen-specific IgE levels were determined in eight bitches at three different time-points of their reproductive cycle: anestrous, estrous and pregnancy. RESULTS: Total IgE was significantly decreased (median: 74%) in female dogs during pregnancy when compared to anestrous. In 14 of 216 (6%), allergen-specific IgE test results were variably positive and negative at different stages of the reproductive cycle. This variation, however, was not related to changes in total serum IgE levels. CONCLUSIONS: Total IgE serum levels are reduced during pregnancy in female dogs. However, results of one allergen-specific IgE test did not appear to be markedly altered by the reproductive cycle in healthy bitches.

Nitric Oxide Mediates Nitrite-Sensing and Acclimation and Triggers a Remodeling of Lipids.

Nitric oxide (NO) is an intermediate of the nitrogen cycle, an industrial pollutant, and a marker of climate change. NO also acts as a gaseous transmitter in a variety of biological processes. The impact of environmental NO needs to be addressed. In diatoms, a dominant phylum in phytoplankton, NO was reported to mediate programmed cell death in response to diatom-derived polyunsaturated aldehydes. Here, using the Phaeodactylum Pt1 strain, 2E,4E-decadienal supplied in the micromolar concentration range led to a nonspecific cell toxicity. We reexamined NO biosynthesis and response in Phaeodactylum NO inhibits cell growth and triggers triacylglycerol (TAG) accumulation. Feeding experiments indicate that NO is not produced from Arg but via conversion of nitrite by the nitrate reductase. Genome-wide transcriptional analysis shows that NO up-regulates the expression of the plastid nitrite reductase and genes involved in the subsequent incorporation of ammonium into amino acids, via both Gln synthesis and Orn-urea pathway. The phosphoenolpyruvate dehydrogenase complex is also up-regulated, leading to the production of acetyl-CoA, which can feed TAG accumulation upon exposure to NO. Transcriptional reprogramming leading to higher TAG content is balanced with a decrease of monogalactosyldiacylglycerol (MGDG) in the plastid via posttranslational inhibition of MGDG synthase enzymatic activity by NO. Intracellular and transient NO emission acts therefore at the basis of a nitrite-sensing and acclimating system, whereas a long exposure to NO can additionally induce a redirection of carbon to neutral lipids and a stress response.

Co-occurrence of Pain Symptoms and Somatosensory Sensitivity in Burning Mouth Syndrome: A Systematic Review.

BACKGROUND: Burning mouth syndrome (BMS) is a chronic and spontaneous oral pain with burning quality in the tongue or other oral mucosa without any identifiable oral lesion or laboratory finding. Pathogenesis and etiology of BMS are still unknown. However, BMS has been associated with other chronic pain syndromes including other idiopathic orofacial pain, the dynias group and the family of central sensitivity syndromes. This would imply that BMS shares common mechanisms with other cephalic and/or extracephalic chronic pains. The primary aim of this systematic review was to determine whether BMS is actually associated with other pain syndromes, and to analyze cephalic and extracephalic somatosensory sensitivity in these patients. METHODS: This report followed the PRISMA Statement. An electronic search was performed until January 2015 in PubMed, Cochrane library, Wiley and ScienceDirect. Searched terms included "burning mouth syndrome OR stomatodynia OR glossodynia OR burning tongue OR oral burning". Studies were selected according to predefined inclusion criteria (report of an association between BMS and other pain(s) symptoms or of cutaneous cephalic and/or extracephalic quantitative sensory testing in BMS patients), and a descriptive analysis conducted. RESULTS: The search retrieved 1512 reports. Out of these, twelve articles met criteria for co-occurring pain symptoms and nine studies for quantitative sensory testing (QST) in BMS patients. The analysis reveals that in BMS patients co-occurring pain symptoms are rare, assessed by only 0.8% (12 of 1512) of the retrieved studies. BMS was associated with headaches, TMD, atypical facial pain, trigeminal neuralgia, post-herpetic facial pain, back pain, fibromyalgia, joint pain, abdominal pain, rectal pain or vulvodynia. However, the prevalence of pain symptoms in BMS patients is not different from that in the age-matched general population. QST studies reveal no or inconsistent evidence of abnormal cutaneous cephalic and extracephalic somatosensory sensitivity. CONCLUSIONS: There is no evidence for a high rate of other pain symptoms or somatosensory impairments co-occurring with BMS. These results thus suggest that BMS rather depends on specific mechanisms, likely at the trigeminal level. Nevertheless, more thoroughly conducted research is required to draw definitive conclusion.

A predominantly neolithic origin for European paternal lineages.

The relative contributions to modern European populations of Paleolithic hunter-gatherers and Neolithic farmers from the Near East have been intensely debated. Haplogroup R1b1b2 (R-M269) is the commonest European Y-chromosomal lineage, increasing in frequency from east to west, and carried by 110 million European men. Previous studies suggested a Paleolithic origin, but here we show that the geographical distribution of its microsatellite diversity is best explained by spread from a single source in the Near East via Anatolia during the Neolithic. Taken with evidence on the origins of other haplogroups, this indicates that most European Y chromosomes originate in the Neolithic expansion. This reinterpretation makes Europe a prime example of how technological and cultural change is linked with the expansion of a Y-chromosomal lineage, and the contrast of this pattern with that shown by maternally inherited mitochondrial DNA suggests a unique role for males in the transition.

Effect of lingual nerve block on burning mouth syndrome (stomatodynia): a randomized crossover trial.

Burning mouth syndrome (stomatodynia) is associated with changes of a neuropathic nature the main location of which, peripheral or central, remains unknown. A randomised, double-blind crossover design was used to investigate the effects of lingual nerve block on spontaneous burning pain and a possible correlation with the effects of topical clonazepam, the patient's response to a psychological questionnaire, and the taste and heat thresholds. The spontaneous burning was measured with a visual analogue scale (VAS) just before and 15 min after injection. The decreases in VAS score after lidocaine or saline injection were not significantly different (2.7+/-3.9 and 2.0+/-2.6, respectively; n=20). However, two groups of patients could be identified: in a "peripheral group" (n=10) the VAS decrease due to lingual nerve injection was 4.3+/-3.1cm after lidocaine and 0.9+/-0.3 cm after saline (p=0.02). In a "central group" (n=7), there were an increase in pain intensity score (-0.8+/-2.6 cm) after lidocaine and a decrease (1.5+/-3.0 cm) after saline (p=0.15). An increase in the hospital anxiety and depression (HAD) score and a decreased taste sensitivity and heat pain threshold of painful oral area were seen in patients compared with age-and-sex-matched controls (p<0.05). Topical clonazepam treatment tended to be more effective (p=0.07) and HAD score lower (p<0.03) in the peripheral than in the central group. These results suggest that the neuropathic disorder associated with stomatodynia may be predominantly peripheral, central or mixed depending on the individual. Topical application of clonazepam and HAD may serve as indicators of which mechanism is dominating.

Catalytic mechanism of Sulfiredoxin from Saccharomyces cerevisiae passes through an oxidized disulfide sulfiredoxin intermediate that is reduced by thioredoxin.

Sulfiredoxin catalyzes the ATP-dependent reduction of overoxidized eukaryotic 2-Cys peroxiredoxin PrxSO(2) into sulfenic PrxSOH. Recent mechanistic studies on sulfiredoxins have validated a catalytic mechanism that includes formation of a phosphoryl intermediate on the sulfinyl moiety of PrxSO(2), followed by an attack of the catalytic cysteine of sulfiredoxin on the phosphoryl intermediate that leads to formation of a thiosulfinate intermediate PrxSO-S-sulfiredoxin. Formation of this intermediate implies the recycling of sulfiredoxin into the reduced form. In this study, we have investigated how the reductase activity of the Saccharomyces cerevisiae sulfiredoxin is regenerated. The results show that an oxidized sulfiredoxin under disulfide state is formed between the catalytic Cys(84) and Cys(48). This oxidized sulfiredoxin species is shown to be catalytically competent along the sulfiredoxin-recycling process and is reduced selectively by thioredoxin. The lack of Cys(48) in the mammalian sulfiredoxins and the low efficiency of reduction of the thiosulfinate intermediate by thioredoxin suggest a recycling mechanism in mammals different from that of sulfiredoxin from Saccharomyces cerevisiae.

Steroid dysregulation and stomatodynia (burning mouth syndrome).

Stomatodynia ( burning mouth syndrome) is characterized by a spontaneous, continuous burning pain felt in the oral mucosa typically of anxiodepressive menopausal women. Because there is no obvious organic cause, it is considered a nonspecific pain. This Focus Article proposes a hypothesis based on the following pathophysiological cascade: chronic anxiety or post traumatic stress leads to a dysregulation of the adrenal production of steroids. One consequence is a decreased or modified production of some major precursors for the neuroactive steroid synthesis occurring in the skin, mucosa, and nervous system. At menopause, the drastic fall of the other main precursor supply , the gonadal steroids, leads to a brisk alteration of the production of neuroactive steroids. This results in neurodegenerative alterations of small nerves fibers of the oral mucosa and /or some brain areas involved in oral somatic sensations. These neuropathic changes become irreversible and precipitate the burning pain, dysgeusia, and xerostomia associated with stomatodynia, which all involve thin nerve fibers.

Evidence on correlation between number of disulfide bridge and toxicity of class IIa bacteriocins.

The aim of this work was to study in the same microbiological conditions and for the first time the inhibitory action of eight class IIa bacteriocins against 23 Gram-positive bacteria in which some of them are industrially used as starters and against the eight class IIa bacteriocins producing strains themselves. The sensitivity of each strain was determined after MPA using the mathematical model described by Cabo et al. (1999, J. Appl. Microbiol. 87, 907-914). Therefore, the principal component analysis realized led to a strong correlation between spectrum and extra-disulfide bridge and a minor correlation between spectrum and isoelectric point of each bacteriocin.

Validation of a French version of the Child-OIDP index.

The Child-OIDP index is an indicator of oral health-related quality of life, which has been validated among 12-yr-old children in Thailand. The aim of this study was to assess the reliability, validity, and applicability of this questionnaire among French children. After translation and cultural adaptation, the Child-OIDP was tested on 414 10-yr-old children in France. The children completed the Child-OIDP in face-to-face interviews, were clinically examined, and answered questions evaluating their global self-rated oral health. Parents filled in a questionnaire concerning their socio-demographic background. An oral impact on daily life was reported by 73% of the children. The mean Child-OIDP score was 6.32 [standard deviation (SD) 8.22] and the median was 3.33. The internal reliability was confirmed with a Cronbach's alpha of 0.57. The retest procedure (n = 62) showed a satisfactory reproducibility (r = 0.81, kappa = 0.75). The index was shown to be a valid instrument. Construct validity was satisfactory as the Child-OIDP score increased when the children's perceived oral health decreased. The Child-OIDP score was able to discriminate between different socio-demographic groupings and varied according to dental status. This study showed that the Child-OIDP is applicable for use among children in France. It has promising psychometric properties but further research is required to evaluate its sensitivity to change.

The egocentric reference deviation of neglect patients is influenced by visuospatial attention.

The right deviation of the subjective straight-ahead (SSA), representing a deviation of the body centered spatial reference frame (egocentric reference), is a frequent phenomenon in spatial neglect. Little is known about the influence of visuo-spatial attention on this SSA shift. The aim of this study was to investigate the influence of eye direction (overt attention) and perception of the visual background (covert attention) on the SSA pointing. We included 12 patients with right hemisphere stroke. Six were classified as neglect (N+) and compared to the six non neglect (N-) patients and 19 normal control subjects (C). They had to point straight-ahead (right hand) on an horizontal board. Pointing was performed in the light or in darkness, first with spontaneous eye direction, then while fixating a visual target (-15 degrees , 0 degrees , +15 degrees ). A first ANOVA of factors group, eye direction (left, center, right fixation) and visual context (light, darkness) showed a right SSA deviation in N+ patients only, which was more severe in the light than in darkness. In this group, the SSA was shifted in the same direction as the target, while that of N- and C groups was mildly shifted in the opposite direction. The comparison of spontaneous and central fixation conditions also showed an eye direction by group interaction, as the spontaneous right SSA deviation was reduced by central fixation in N+ patients only. These results suggest that, in neglect patients, the egocentric reference deviation is not a fixed phenomenon, and that it can be influenced by manipulation of both overt and covert spatial attention.

Towards a new taxonomy of idiopathic orofacial pain.

There is no current consensus on the taxonomy of the different forms of idiopathic orofacial pain (stomatodynia, atypical odontalgia, atypical facial pain, facial arthromyalgia), which are sometimes considered as separate entities and sometimes grouped together. In the present prospective multicentric study, we used a systematic approach to help to place these different painful syndromes in the general classification of chronic facial pain. This multicenter study was carried out on 245 consecutive patients presenting with chronic facial pain (>4 months duration). Each patient was seen by two experts who proposed a diagnosis, administered a 111-item questionnaire and filled out a standardized 68-item examination form. Statistical processing included univariate analysis and several forms of multidimensional analysis. Migraines (n=37), tension-type headache (n=26), post-traumatic neuralgia (n=20) and trigeminal neuralgia (n=13) tended to cluster independently. When signs and symptoms describing topographic features were not included in the list of variables, the idiopathic orofacial pain patients tended to cluster in a single group. Inside this large cluster, only stomatodynia (n=42) emerged as a distinct homogenous subgroup. In contrast, facial arthromyalgia (n=46) and an entity formed with atypical facial pain (n=25) and atypical odontalgia (n=13) could only be individualised by variables reflecting topographical characteristics. These data provide grounds for an evidence-based classification of idiopathic facial pain entities and indicate that the current sub-classification of these syndromes relies primarily on the topography of the symptoms.

The expression and function of the endothelin system in contractile properties of vaginal myofibroblasts of women with uterovaginal prolapse.

OBJECTIVE: The endothelin-1 system regulates (myo)fibroblast contraction in wound healing. Our aim was to determine endothelin-1 system expression and function in contractile properties of vaginal myofibroblasts of women with uterovaginal prolapse. STUDY DESIGN: Cultures of alpha-smooth muscle actin-positive myofibroblasts that were established at the time of repair surgery for prolapse (n = 30; mean age, 56 +/- 14 years) were analyzed and compared for their expression of the endothelin-1 system and contractile properties to myofibroblasts from primiparous women. RESULTS: Myofibroblasts expressed the complete endothelin system but did not secrete endothelin-1. Endothelin-1 binding was mediated exclusively by the endothelin B-receptor. In 3-dimensional collagen gels, spontaneous contraction of myofibroblasts from estrogen-treated women with prolapse was statistically significantly lower than from young primiparous women. Exogenous addition of endothelin-1 decreased the spontaneous contraction of myofibroblasts. CONCLUSION: Genital myofibroblasts of women with uterovaginal prolapse are poorly contractile, and endothelin-1 further decreases vaginal myofibroblast contraction, which is opposite to observations in skin myofibroblasts.

Straight ahead in spatial neglect: evidence that space is shifted, not rotated.

The egocentric reference appears to be altered in neglect patients, as evidenced by an ipsilesional error when pointing straight ahead. In this study, patients with right brain damage used a horizontal rod to show this straight-ahead direction. There was a systematic rightward translation of the rod, and no clockwise rotation occurred in five of six neglect patients. The results suggest a preferential rightward translation of space representation in neglect.