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Carbohydrates have various functions in biological systems. However, the structural analysis of carbohydrates remains challenging. Most of the commonly used methods involve derivatization of carbohydrates or can only identify part of the structure. Here, we report a de novo method for completely structural identification of underivatised oligosaccharides. This method, which can provide assignments of linkages, anomeric configurations, and branch locations, entails low-energy collision-induced dissociation (CID) of sodium ion adducts that enable the cleavage of selective chemical bonds, a logical procedure to identify structurally decisive fragment ions for subsequent CID, and the specially prepared disaccharide CID spectrum databases. This method was first applied to determine the structures of four underivatised glucose oligosaccharides. Then, high-performance liquid chromatography and a mass spectrometer with a built-in logical procedure were established to demonstrate the capability of the in situ CID spectrum measurement and structural determination of the oligosaccharides in chromatogram. This consolidation provides a simple, rapid, sensitive method for the structural determination of glucose oligosaccharides, and applications to oligosaccharides containing hexoses other than glucose can be made provided the corresponding disaccharide databases are available.




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Several advancements in the de novo structural identification of monosaccharides and oligosaccharides have been demonstrated recently. Nagy et al. reported a fixed-ligand kinetic method for the determination of monosaccharide absolute configuration32. However, this method was not used for the determination of the linkage positions, anomeric configurations, sequences, and branch locations of oligosaccharides. Konda et al. reported that the CID of anion m/z 163 exhibited distinct fragmentation fingerprints corresponding to linkage positions. They applied the CID of anion m/z 163 to the linkage determination of 18O-labelled linear oligosaccharides33. Bendiak et al. demonstrated that anion m/z 221 can be used to identify the stereochemistry and anomeric configuration of hexose in oligosaccharides34,35,36. This method has the following limitations: The reducing end must be derivatised, resulting in the structure of two hexoses on the reducing side cannot be determined, anion intensities are usually low that it may take several hours to obtain a mass spectrum with a good signal-to-noise ratio, a complicated mass spectrometer is required, and this method is currently used for only linear oligosaccharides.


Recently we have proposed a new de novo method for determining the entire structure of underivatised oligosaccharides through CID tandem MS of sodium ion adducts37. In this study, the structural determination of glucose trisaccharides and tetrasaccharides was demonstrated. This method can be extended to larger oligosaccharides and oligosaccharides containing hexoses other than glucose.


De novo root organogenesis is the process in which adventitious roots regenerate from detached or wounded plant tissues or organs. In tissue culture, appropriate types and concentrations of plant hormones in the medium are critical for inducing adventitious roots. However, in natural conditions, regeneration from detached organs is likely to rely on endogenous hormones. To investigate the actions of endogenous hormones and the molecular mechanisms guiding de novo root organogenesis, we developed a simple method to imitate natural conditions for adventitious root formation by culturing Arabidopsis thaliana leaf explants on B5 medium without additive hormones. Here we show that the ability of the leaf explants to regenerate roots depends on the age of the leaf and on certain nutrients in the medium. Based on these observations, we provide examples of how this method can be used in different situations, and how it can be optimized. This simple method could be used to investigate the effects of various physiological and molecular changes on the regeneration of adventitious roots. It is also useful for tracing cell lineage during the regeneration process by differential interference contrast observation of β-glucuronidase staining, and by live imaging of proteins labeled with fluorescent tags.


Mechanisms of de novo mutations. De novo mutations can arise because of static properties of the genome, such as the underlying sequence (deamination of methylated CpGs, transitions versus transversions) or due to erroneous pairing of nucleotides during DNA replication. However, de novo mutations can also occur in relation to cell-specific properties such as the chromatin state, transcriptional status, and gene expression levels. Mutational hotspots for genomic rearrangements are largely determined by the underlying genomic architecture. One such example is given for non-allelic homologous recombination (NAHR). Arrows represent the influence of each feature on the de novo mutation rate. Green arrows pointing upwards indicate elevated mutability; red arrows pointing downwards indicate lower mutability. M methyl group modifying cytosine


In this review, we first touch on the biological aspects of de novo mutations in humans, such as their origin, distribution throughout the genome, and factors related to their occurrence and timing. Later, we discuss the increasingly recognized role of de novo mutations in human disease and other translational aspects. Throughout, we will focus mostly on de novo SNVs; readers should refer to Box 2 and previous work from others for more information on the role of de novo CNVs and other structural genomic variation in human disease [36, 37].


The difference between the rate at which pre-mutagenic damage appears in DNA and the rate at which it is repaired defines the rate at which de novo mutations arise. It is often assumed that germline de novo mutations originate from errors in DNA replication during gametogenesis, particularly in sperm cells and their precursors (see section below on parental origin of de novo mutations). However, inefficient repair of spontaneous DNA lesions can also give rise to de novo mutations during spermatogenesis, as continuous proliferation and short periods between cell divisions can translate into there being less time to repair these lesions [44, 45]. Furthermore, in oogenesis, spontaneous DNA mutations coupled to inefficient repair mechanisms might play a more prominent role [44]. Therefore, while the de novo mutation rate is a reflection of the replication error rate and the number of mitoses a cell has undergone, this number is also influenced by the amount of time between mitoses and the efficiency of the DNA repair [44].


Another origin for some of these clusters could be chromosomal rearrangements. It has been shown that the mutation rate for SNVs is elevated and SNVs can cluster in proximity to the breakpoints of de novo CNVs [62, 63]. This is likely the result of the replicative CNV mechanism in which a low-fidelity, error-prone DNA polymerase is used during repair of DNA. Indeed, work performed in yeast supports the observation that double-strand-break-induced replication is a source of mutation clusters [61].


In contrast to the mutation clusters that occur within one individual, mutational hotspots are considered overlapping loci that are found to be mutated more frequently than expected in different individuals. Recent research based on WGS datasets and modeling has identified such hotspots in coding sequences [9]. Furthermore, the existence of these mutational hotspots has been recently confirmed in a larger study that showed specific bins of 1 Mb within the human genome with elevated mutation rates [13]. Interestingly, in this study, two bins including genes CSMD1 and WWOX were shown to have a higher maternal than paternal mutation rate. The mechanism for this is still largely unknown, but the latter is a well-known fragile site within the human genome [64]. Other sites of the human genome that are especially prone to de novo mutations include ribosomal DNA (rDNA) gene clusters [65], segmental duplications [66], and microsatellites [67], with mutation rates three to four orders of magnitude higher than average [68].


Approximately 80% of all de novo germline point mutations arise on the paternal allele, and advanced paternal age at conception has been established as the major factor linked to the increase in the number of de novo mutations in the offspring, both at the population level and within the same family (Fig. 2) [11, 13, 15]. Spermatogonial cells continue to divide throughout life, which is likely to allow the progressive accumulation of mutations due to errors during DNA replication but also as a result of failure to repair non-replicative DNA damage between cell divisions [44]. Furthermore, the efficiency of endogenous defense systems against radical oxygen species and of DNA repair mechanisms might also decline with age [71, 72]. De novo mutations in children of young fathers show a different signature and localize to later-replicating regions of the genome compared with those of children of old fathers, suggesting that additional factors contribute to de novo mutations with age [12, 13]. It has been calculated that one to three de novo mutations are added to the germline mutational load of the offspring for each paternal year at conception, but this effect varies considerably between families [11, 13]. This variability has been suggested to be due to individual differences in the rate of mutagenesis, in the frequency of spermatogonial stem cell division and even to genetic variation in DNA mismatch repair genes [11]. Indeed, one could speculate that deleterious variation in genes involved in replication and repair could predispose to elevated de novo mutation rates not only in somatic cells but also in the germline, as has been observed in mouse models lacking exonuclease activity in DNA polymerase δ [73]. 350c69d7ab


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