Effects of specific disease mutations in non-muscle myosin 2A on its structure and function.

Non-muscle myosin 2A (NM2A), a widely expressed class 2 myosin, is important for organizing actin filaments in cells. It cycles between a compact inactive 10S state in which its regulatory light chain (RLC) is dephosphorylated and a filamentous state in which the myosin heads interact with actin, and the RLC is phosphorylated. Over 170 missense mutations in MYH9, the gene that encodes the NM2A heavy chain, have been described. These cause MYH9 disease, an autosomal-dominant disorder that leads to bleeding disorders, kidney disease, cataracts, and deafness. Approximately two-thirds of these mutations occur in the coiled-coil tail. These mutations could destabilize the 10S state and/or disrupt filament formation or both. To test this, we determined the effects of six specific mutations using multiple approaches, including circular dichroism to detect changes in secondary structure, negative stain electron microscopy to analyze 10S and filament formation in vitro, and imaging of GFP-NM2A in fixed and live cells to determine filament assembly and dynamics. Two mutations in D1424 (D1424G and D1424N) and V1516M strongly decrease 10S stability and have limited effects on filament formation in vitro. In contrast, mutations in D1447 and E1841K, decrease 10S stability less strongly but increase filament lengths in vitro. The dynamic behavior of all mutants was altered in cells. Thus, the positions of mutated residues and their roles in filament formation and 10S stabilization are key to understanding their contributions to NM2A in disease.

The ethylene-responsive transcription factor ERF024 is a novel regulator of climacteric fruit ripening in melon.

Fruit ripening is an essential developmental stage in Angiosperms triggered by hormonal signals such as ethylene, a major player in climacteric ripening. Melon is a unique crop showing both climacteric and non-climacteric cultivars, offering an ideal model for dissecting the genetic mechanisms underpinning this process. The major quantitative trait locus ETHQV8.1 was previously identified as a key regulator of melon fruit ripening. Here, we narrowed down ETHQV8.1 to a precise genomic region containing a single gene, the transcription factor CmERF024. Functional validation using CRISPR/Cas9 knock-out plants unequivocally identified CmERF024 as the causal gene governing ETHQV8.1. The erf024 mutants exhibited suppression of ethylene production, leading to a significant delay and attenuation of fruit ripening. Integrative multi-omic analyses encompassing RNA-seq, DAP-seq, and DNase-seq revealed the association of CmERF024 with chromatin accessibility and gene expression dynamics throughout fruit ripening. Our data suggest CmERF024 as a novel regulator of climacteric fruit ripening in melon.

Regulation of climacteric fruit ripening in melon: recent advances and future challenges.

Fruit ripening is a complex and highly regulated process where tomato and strawberry have been the model species classically used for studying climacteric and non-climacteric fleshy fruit ripening types, respectively. Melon has emerged as an alternative ripening model because climacteric and non-climacteric cultivars exist, which makes it possible to dissect the regulation of ripening using a genetic approach. Several quantitative trait loci that regulate climacteric fruit ripening have been identified to date, and their combination in both climacteric and non-climacteric genetic backgrounds resulted in lines with different ripening behaviors, demonstrating that the climacteric intensity can be genetically modulated. This review discusses our current knowledge of the physiological changes observed during melon climacteric fruit ripening such as ethylene production, fruit abscission, chlorophyll degradation, firmness, and aroma, as well as their complex genetic control. From pioneer experiments in which ethylene biosynthesis was silenced, to the recent genetic edition of ripening regulators, current data suggest that the climacteric response is determined by the interaction of several loci under quantitative inheritance. The exploitation of the rich genetic diversity of melon will enable the discovery of additional genes involved in the regulation of the climacteric response, ultimately leading to breeding aromatic melon fruits with extended shelf life.

GENOMES UNCOUPLED1-independent retrograde signaling targets the ethylene pathway to repress photomorphogenesis.

When germinating in the light, Arabidopsis (Arabidopsis thaliana) seedlings undergo photomorphogenic development, characterized by short hypocotyls, greening, and expanded cotyledons. Stressed chloroplasts emit retrograde signals to the nucleus that induce developmental responses and repress photomorphogenesis. The nuclear targets of these retrograde signals are not yet fully known. Here, we show that lincomycin-treated seedlings (which lack developed chloroplasts) show strong phenotypic similarities to seedlings treated with ethylene (ET) precursor 1-aminocyclopropane-1-carboxylic acid, as both signals inhibit cotyledon separation in the light. We show that the lincomycin-induced phenotype partly requires a functioning ET signaling pathway, but could not detect increased ET emissions in response to the lincomycin treatment. The two treatments show overlap in upregulated gene transcripts, downstream of transcription factors ETHYLENE INSENSITIVE3 and EIN3-LIKE1. The induction of the ET signaling pathway is triggered by an unknown retrograde signal acting independently of GENOMES UNCOUPLED1. Our data show how two apparently different stress responses converge to optimize photomorphogenesis.

CRISPR/Cas9 gene editing uncovers the roles of CONSTITUTIVE TRIPLE RESPONSE 1 and REPRESSOR OF SILENCING 1 in melon fruit ripening and epigenetic regulation.

Melon (Cucumis melo) has emerged as an alternative model to tomato for studying fruit ripening due to the coexistence of climacteric and non-climacteric varieties. Previous characterization of a major quantitative trait locus (QTL), ETHQV8.1, that is able to trigger climacteric ripening in a non-climacteric background resulted in the identification of a negative regulator of ripening CTR1-like (MELO3C024518) and a putative DNA demethylase ROS1 (MELO3C024516) that is the orthologue of DML2, a DNA demethylase that regulates fruit ripening in tomato. To understand the role of these genes in climacteric ripening, in this study we generated homozygous CRISPR knockout mutants of CTR1-like and ROS1 in a climacteric genetic background. The climacteric behavior was altered in both loss-of-function mutants in two growing seasons with an earlier ethylene production profile being observed compared to the climacteric wild type, suggesting a role of both genes in climacteric ripening in melon. Single-cytosine methylome analyses of the ROS1-knockout mutant revealed changes in DNA methylation in the promoter regions of the key ripening genes such as ACS1, ETR1, and ACO1, and in transcription factors associated with ripening including NAC-NOR, RIN, and CNR, suggesting the importance of ROS1-mediated DNA demethylation for triggering fruit ripening in melon.

Life with a stoma across five European countries-a cross-sectional study on long-term rectal cancer survivors.

PURPOSE: Stoma-related problems are known to be important to patients and potentially affect everyday life. The prevalence of stoma-related problems in rectal cancer survivors remains undetermined. This study aimed to examine aspects of life with a long-term stoma, stoma management, and stoma-related problems and explore the impact of stoma-related problems on daily life. METHODS: In total, 2262 patients from 5 European countries completed a multidimensional survey. Stoma-related problems were assessed using the Colostomy Impact score. Multivariable regression analysis, after adjusting for potential confounding factors, provided odds ratio (OR) and 95% confidence intervals (CI) for stoma-related problems' association with restrictions in daily life. RESULTS: The 2262 rectal cancer survivors completed the questionnaire at a median of 5.4 years (interquartile range 3.8-7.6) after stoma formation. In the total sample, leakage (58%) and troublesome odour (55%) were most prevalent followed by skin problems (27%) and pain (21%). Stoma-related problems were more prevalent in patients with parastomal bulging. A total of 431 (19%) reported feeling restricted in daily activities in life with a stoma. Leakage, odour, skin problems, stool consistency, and frequent appliance changes were significantly associated with restrictions in daily life. The highest risk of experiencing restrictions was seen for patients having odour (OR 2.74 [95% CI: 1.99-3.78]) more than once a week and skin problems (OR 1.77 [95% CI: 1.38-2.27]). CONCLUSION: In this large cohort with rectal cancer, stoma-related problems were highly prevalent and impacted daily life. Supportive care strategies should entail outreach to patients with a long-term stoma.

Translation and international validation of the Colostomy Impact score.

AIM: Optimal oncological resection in cancers of the lower rectum often requires a permanent colostomy. However, in some patients a colostomy may have a negative impact on health-related quality of life (HRQoL). The Colostomy Impact (CI) score is a simple questionnaire that identifies patients with stoma dysfunction that impairs HRQoL by dividing patients into 'minor' and 'major' CI groups. This aim of this study is to evaluate construct and discriminative validity, sensitivity, specificity and reliability of the CI score internationally, making it applicable for screening and identification of patients with stoma-related impaired HRQoL. METHOD: The CI score was translated in agreement with WHO recommendations. Cross-sectional cohorts of rectal cancer survivors with a colostomy in Australia, China, Denmark, the Netherlands, Portugal, Spain and Sweden were asked to complete the CI score, the European Organization for Research and Treatment of Cancer (EORTC) quality of life 30-item core questionnaire, the stoma-specific items of the EORTC quality of life 29-item colorectal-specific questionnaire and five anchor questions assessing the impact of colostomy on HRQoL. RESULTS: A total of 2470 patients participated (response rate 51%-93%). CI scores were significantly higher in patients reporting reduced HRQoL due to their colostomy than in patients reporting no reduction. Differences in EORTC scale scores between patients with minor and major CI were significant and clinically relevant. Sensitivity was high regarding dissatisfaction with a colostomy. Regarding evaluation of discriminative validity, the CI score relevantly identified groups with differences in HRQoL. The CI score proved reliable, with equal CI scores between test and retest and an intraclass correlation coefficient in the moderate to excellent range. CONCLUSION: The CI score is internationally valid and reliable. We encourage its use in clinical practice to identify patients with stoma dysfunction who require further attention.

Prediction of advanced colonic neoplasm in symptomatic patients: a scoring system to prioritize colonoscopy (COLONOFIT study).

BACKGROUND: Fast-track colonoscopy to detect patients with colorectal cancer based on high-risk symptoms is associated with low sensitivity and specificity. The aim was to derive a predictive score of advanced colonic neoplasia in symptomatic patients in fast-track programs. METHODS: All patients referred for fast-track colonoscopy were evaluated. Faecal immunological haemoglobin test (3 samples; positive> 4 µg Hb/g), and a survey to register clinical variables of interest were performed. Colorectal cancer and advanced adenoma were considered as advanced colonic neoplasia. A sample size of 600 and 500 individuals were calculated for each phase 1 and phase 2 of the study, respectively (Phase 1, derivation and Phase 2, validation cohort). A Bayesian logistic regression analysis was used to derive a predictive score. RESULTS: 1495 patients were included. Age (OR, 21), maximum faecal-Hb value (OR, 2.3), and number of positive samples (OR, 28) presented the highest ORs predictive of advanced colonic neoplasia. The additional significant predictive variables adjusted for age and faecal-Hb variables in Phase 1 were previous colonoscopy (last 5 years) and smoking (no, ex/active). With these variables a predictive score of advanced colonic neoplasia was derived. Applied to Phase 2, patients with a Score > 20 had an advanced colonic neoplasia probability of 66% (colorectal cancer, 32%), while those with a Score ≤ 10, a probability of 10% (colorectal cancer, 1%). Prioritizing patients with Score > 10, 49.4% of patients would be referred for fast-track colonoscopy, diagnosing 98.3% of colorectal cancers and 77% of advanced adenomas. CONCLUSIONS: A scoring system was derived and validated to prioritize fast-track colonoscopies according to risk, which was efficient, simple, and robust.

Non-invasive quantification of ethylene in attached fruit headspace at 1 p.p.b. by gas chromatography-mass spectrometry.

Ethylene is a gaseous plant hormone involved in defense, adaptations to environmental stress and fruit ripening. Its relevance to the latter makes its detection highly useful for physiologists interested in the onset of ripening. Produced as a sharp peak during the respiratory burst, ethylene is biologically active at tens of nl L-1 . Reliable quantification at such concentrations generally requires specialized instrumentation. Here we present a rapid, high-sensitivity method for detecting ethylene in attached fruit using a conventional gas chromatography-mass spectrometry (GC-MS) system and in situ headspace collection chambers. We apply this method to melon (Cucumis melo L.), a unique species consisting of climacteric and non-climacteric varieties, with a high variation in the climacteric phenotype among climacteric types. Using a population of recombinant inbred lines (RILs) derived from highly climacteric ('Védrantais', cantalupensis type) and non-climacteric ('Piel de Sapo', inodorus type) parental lines, we observed a significant variation for the intensity, onset and duration of the ethylene burst during fruit ripening. Our method does not require concentration, sampling times over 1 h or fruit harvest. We achieved a limit of detection of 0.41 ± 0.04 nl L-1 and a limit of quantification of 1.37 ± 0.13 nl L-1 with an analysis time per sample of 2.6 min. Validation of the analytical method indicated that linearity (>98%), precision (coefficient of variation ≤2%) and sensitivity compared favorably with dedicated optical sensors. This study adds to evidence of the characteristic climacteric ethylene burst as a complex trait whose intensity in our RIL population lies along a continuum in addition to two extremes.

Interplay of Endocytosis and Growth Factor Receptor Signalling.

Growth factor receptors play a variety of roles during embryonic development and in adult homeostasis. These receptors are activated repeatedly in different cellular contexts and with different cellular outcomes. This begs the question as to how cells in a particular developmental, spatial and temporal context, or in adult tissue, interpret signalling by growth factor receptors in order to deliver qualitatively different signalling outputs. One mechanism by which this could occur is via endocytic regulation. The original paradigm for the role of endocytosis in growth factor receptor signalling was that receptor uptake has a quantitative role in signalling by reducing the number of cell surface receptors available for activation and targeting activated receptors for degradation. However, a range of studies over the last several years, in many different experimental systems, has demonstrated an additional qualitative role for endocytic trafficking in receptor signalling, with specific outcomes depending on the location of the signalling complex. Confinement of receptors within endosomes can spatially regulate signalling, facilitating specific protein interactions or post-translational modifications that alter throughout the trafficking process. Therefore, endocytosis does not simply regulate cell surface expression, but tightly controls protein interactions and function to produce distinct outcomes.

A study to evaluate the effect of ultrasound treatment on nodules in multiple sclerosis patients.

Purpose/aim: Ultrasound has demonstrated anti-inflammatory and pain-relief benefits in several conditions such as cellulite or trauma events. We assessed the efficacy of ultrasound therapy on nodules associated with first-line treatments in multiple sclerosis patients. MATERIALS AND METHODS: Twenty-two multiple sclerosis patients were enrolled during 2013 and randomized to two groups: in the control group patients were treated only with a conventional gel prescribed for cellulite and nodules, while in the experimental group the gel was combined with ultrasound therapy. Patients were treated during 10 weeks and followed up for 10 additional weeks. Three nodules were assessed for each patient, measuring size, pain and redness at 0, 10 and 20 weeks. RESULTS: We found a significant decrease in both groups in size, pain and redness across the three visits (p < 0.0001 for size, p = 0.01 and p < 0.0001 for pain, and p = 0.0002 and p < 0.0001 for redness, respectively for the difference at visit 2 and 3 with respect to visit 1). More interestingly, we observed a greater reduction in pain and redness in the ultrasound-treated group, but the difference was only statistically significant at 10 weeks (p = 0.01 for both pain and redness). On the third visit, no differences between control and experimental groups were detected, both achieving the same levels in measured variables. CONCLUSIONS: Both treatments are useful to improve skin reaction after first-line treatments, but ultrasound in combination with gel achieves a faster reduction in pain and redness, suggesting that ultrasound treatment might be a good analgesic for nodule management in multiple sclerosis patients.

Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly.

BACKGROUND: The genome of the melon (Cucumis melo L.) double-haploid line DHL92 was recently sequenced, with 87.5 and 80.8% of the scaffold assembly anchored and oriented to the 12 linkage groups, respectively. However, insufficient marker coverage and a lack of recombination left several large, gene rich scaffolds unanchored, and some anchored scaffolds unoriented. To improve the anchoring and orientation of the melon genome assembly, we used resequencing data between the parental lines of DHL92 to develop a new set of SNP markers from unanchored scaffolds. RESULTS: A high-resolution genetic map composed of 580 SNPs was used to anchor 354.8 Mb of sequence, contained in 141 scaffolds (average size 2.5 Mb) and corresponding to 98.2% of the scaffold assembly, to the 12 melon chromosomes. Over 325.4 Mb (90%) of the assembly was oriented. The genetic map revealed regions of segregation distortion favoring SC alleles as well as recombination suppression regions coinciding with putative centromere, 45S, and 5S rDNA sites. New chromosome-scale pseudomolecules were created by incorporating to the previous v3.5 version an additional 38.3 Mb of anchored sequence representing 1,837 predicted genes contained in 55 scaffolds. Using fluorescent in situ hybridization (FISH) with BACs that produced chromosome-specific signals, melon chromosomes that correspond to the twelve linkage groups were identified, and a standardized karyotype of melon inbred line T111 was developed. CONCLUSIONS: By utilizing resequencing data and targeted SNP selection combined with a large F2 mapping population, we significantly improved the quantity of anchored and oriented melon scaffold genome assembly. Using genome information combined with FISH mapping provided the first cytogenetic map of an inodorus melon type. With these results it was possible to make inferences on melon chromosome structure by relating zones of recombination suppression to centromeres and 45S and 5S heterochromatic regions. This study represents the first steps towards the integration of the high-resolution genetic and cytogenetic maps with the genomic sequence in melon that will provide more information on genome organization and allow for the improvement of the melon genome draft sequence.

Combined use of genetic and genomics resources to understand virus resistance and fruit quality traits in melon.

The availability of the genome sequence of many crop species during the past few years has opened a new era in plant biology, allowing for the performance of massive genomic studies in plant species other than the classical models Arabidopsis and rice. One of these crop species is melon (Cucumis melo), a cucurbit of high economic value that has become an interesting model for the study of biological processes such as fruit ripening, sex determination and phloem transport. The recent availability of the melon genome sequence, together with a number of genetic and genomic resources, provides powerful tools that can be used to assist in the main melon breeding targets, namely disease resistance and fruit quality. In this review, we will describe recent data obtained combining the use of a melon near isogenic line (NIL) population and genomic resources to gain insight into agronomically important traits as fruit ripening, resistance to Cucumber Mosaic virus (CMV) and the accumulation of sugars in fruits.

Genetic regulation of volatile production in two melon introgression line collections with contrasting ripening behavior.

The importance of melon aroma in determining fruit quality has been highlighted in recent years. The fruit volatile profile is influenced by the type of fruit ripening. Non-climacteric fruits contain predominantly aldehydes, while climacteric fruits mainly produce esters. Several genes have been described to participate in volatile organic compounds (VOCs) biosynthesis pathways, but knowledge in this area is still incomplete. In this work we analysed the volatile profile of two reciprocal Introgression Line (IL) collections generated from a cross between 'Piel de Sapo' (PS) and 'Védrantais' (VED) melons, differing in their aroma profile and ripening behaviour. SPME GC-MS was performed to identify genes responsible for VOCs formation. More than 1000 QTLs for many volatiles were detected taken together both populations. Introgressions on chromosomes 3, 5, 6, 7 and 8 modified ester-aldehyde balance and were correlated to ripening changes in both genetic backgrounds. Some previously identified QTLs for fruit ripening might be involved in these phenotypes, such as ETHQV8.1 on chromosome 8 and ETHQV6.3 on chromosome 6. PS alleles on chromosomes 2, 6, 10 and 11 were found to increase ester content when introgressed in VED melons. Terpenes showed to be affected by several genomic regions not related to ripening. In addition, several candidate genes have been hypothesized to be responsible for some of the QTLs detected. The analysis of volatile compounds in two reciprocal IL collections has increased our understanding of the relationship between ripening and aroma and offers valuable plant material to improve food quality in melon breeding programs.

Analysis of mRNA Subcellular Distribution in Collective Cell Migration.

The movement of groups of cells by collective cell migration requires division of labor between group members. Therefore, distinct cell identities, unique cell behaviors, and specific cellular roles are acquired by cells undergoing collective movement. A key driving force behind the acquisition of discrete cell states is the precise control of where, when, and how genes are expressed, both at the subcellular and supracellular level. Unraveling the mechanisms underpinning the spatiotemporal control of gene expression in collective cell migration requires not only suitable experimental models but also high-resolution imaging of messenger RNA and protein localization during this process. In recent times, the highly stereotyped growth of new blood vessels by sprouting angiogenesis has become a paradigm for understanding collective cell migration, and consequently this has led to the development of numerous user-friendly in vitro models of angiogenesis. In parallel, single-molecule fluorescent in situ hybridization (smFISH) has come to the fore as a powerful technique that allows quantification of both RNA number and RNA spatial distribution in cells and tissues. Moreover, smFISH can be combined with immunofluorescence to understand the precise interrelationship between RNA and protein distribution. Here, we describe methods for use of smFISH and immunofluorescence microscopy in in vitro angiogenesis models to enable the investigation of RNA and protein expression and localization during endothelial collective cell migration.

NOD mouse dorsal root ganglia display morphological and gene expression defects before and during autoimmune diabetes development.

Introduction: During the development of Autoimmune Diabetes (AD) an autoimmune attack against the Peripheral Nervous System occurs. To gain insight into this topic, analyses of Dorsal Root Ganglia (DRG) from Non-Obese Diabetic (NOD) mice were carried out. Methods: Histopathological analysis by electron and optical microscopy in DRG samples, and mRNA expression analyzes by the microarray technique in DRG and blood leukocyte samples from NOD and C57BL/6 mice were performed. Results: The results showed the formation of cytoplasmic vacuoles in DRG cells early in life that could be related to a neurodegenerative process. In view of these results, mRNA expression analyses were conducted to determine the cause and/or the molecules involved in this suspected disorder. The results showed that DRG cells from NOD mice have alterations in the transcription of a wide range of genes, which explain the previously observed alterations. In addition, differences in the transcription genes in white blood cells were also detected. Discussion: Taken together, these results indicate that functional defects are not only seen in beta cells but also in DRG in NOD mice. These results also indicate that these defects are not a consequence of the autoimmune process that takes place in NOD mice and suggest that they may be involved as triggers for its development.

Mutual modulation of gut microbiota and the immune system in type 1 diabetes models.

The transgenic 116C-NOD mouse strain exhibits a prevalent Th17 phenotype, and reduced type 1 diabetes (T1D) compared to non-obese diabetic (NOD) mice. A cohousing experiment between both models revealed lower T1D incidence in NOD mice cohoused with 116C-NOD, associated with gut microbiota changes, reduced intestinal permeability, shifts in T and B cell subsets, and a transition from Th1 to Th17 responses. Distinct gut bacterial signatures were linked to T1D in each group. Using a RAG-2-/- genetic background, we found that T cell alterations promoted segmented filamentous bacteria proliferation in young NOD and 116C-NOD, as well as in immunodeficient NOD.RAG-2-/- and 116C-NOD.RAG-2-/- mice across all ages. Bifidobacterium colonization depended on lymphocytes and thrived in a non-diabetogenic environment. Additionally, 116C-NOD B cells in 116C-NOD.RAG-2-/- mice enriched the gut microbiota in Adlercreutzia and reduced intestinal permeability. Collectively, these results indicate reciprocal modulation between gut microbiota and the immune system in rodent T1D models.

Human skeletal myopathy myosin mutations disrupt myosin head sequestration.

Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyze the effects of common MYH7 and MYH2 mutations in the light meromyosin (LMM) region of myosin. Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in silico modeling showed that myosin coiled coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients and fluorescent ATP analog chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with x-ray diffraction measurements to estimate myosin head order, we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofiber mechanics experiments to investigate contractile function showed that myofiber contractility was not affected. These findings indicate that the structural remodeling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies.

Kinetics and mechanism of the racemic addition of trimethylsilyl cyanide to aldehydes catalysed by Lewis bases.

The mechanism by which four Lewis bases, triethylamine, tetrabutylammonium thiocyanate, tetrabutylammonium azide and tetrabutylammonium cyanide, catalyse the addition of trimethylsilyl cyanide to aldehydes is studied by a combination of kinetic and spectroscopic methods. The reactions can exhibit first or second order kinetics corresponding to three different reaction mechanisms. Spectroscopic evidence for the formation of hypervalent silicon species is obtained for reaction between all of the tetrabutylammonium salts and trimethylsilyl cyanide. The reactions are accelerated by the presence of water in the reaction mixture, an effect which is due to a change in the reaction mechanism from Lewis to Brønsted base catalysis. Tetrabutylammonium thiocyanate is shown to be an excellent catalyst for the synthesis of cyanohydrin trimethylsilyl ethers on a preparative scale.

Knock-Out of CmNAC-NOR Affects Melon Climacteric Fruit Ripening.

Fruit ripening is an important process that affects fruit quality. A QTL in melon, ETHQV6.3, involved in climacteric ripening regulation, has been found to be encoded by CmNAC-NOR, a homologue of the tomato NOR gene. To further investigate CmNAC-NOR function, we obtained two CRISPR/Cas9-mediated mutants (nor-3 and nor-1) in the climacteric Védrantais background. nor-3, containing a 3-bp deletion altering the NAC domain A, resulted in ~8 days delay in ripening without affecting fruit quality. In contrast, the 1-bp deletion in nor-1 resulted in a fully disrupted NAC domain, which completely blocked climacteric ripening. The nor-1 fruits did not produce ethylene, no abscission layer was formed and there was no external color change. Additionally, volatile components were dramatically altered, seeds were not well developed and flesh firmness was also altered. There was a delay in fruit ripening with the nor-1 allele in heterozygosis of ~20 days. Our results provide new information regarding the function of CmNAC-NOR in melon fruit ripening, suggesting that it is a potential target for modulating shelf life in commercial climacteric melon varieties.