Search

Approximately 13% of the human genome can fold into non-canonical (non-B) DNA structures (e.g. G-quadruplexes, Z-DNA, etc.), which have been implicated in vital cellular processes. Non-B DNA also hinders replication, increasing errors and facilitating mutagenesis, yet its contribution to genome-wide variation in mutation rates remains unexplored. Here, we conducted a comprehensive analysis of nucleotide substitution frequencies at non-B DNA loci within noncoding, non-repetitive genome regions, their ±2 kb flanking regions, and 1-Megabase windows, using human-orangutan divergence and human single-nucleotide polymorphisms. Functional data analysis at single-base resolution demonstrated that substitution frequencies are usually elevated at non-B DNA, with patterns specific to each non-B DNA type. Mirror, direct and inverted repeats have higher substitution frequencies in spacers than in repeat arms, whereas G-quadruplexes, particularly stable ones, have higher substitution frequencies in loops than in stems. Several non-B DNA types also affect substitution frequencies in their flanking regions. Finally, non-B DNA explains more variation than any other predictor in multiple regression models for diversity or divergence at 1-Megabase scale. Thus, non-B DNA substantially contributes to variation in substitution frequencies at small and large scales. Our results highlight the role of non-B DNA in germline mutagenesis with implications to evolution and genetic diseases., Graphical Abstract Graphical AbstractGuiblet et al. show that loci capable of forming non-canonical (non-B) DNA structures are a major driver of variation in nucleotide substitution levels across the genome. Image credit: Wilfried Guiblet.

DNA conformation may deviate from the classical B-form in ~13% of the human genome. Non-B DNA regulates many cellular processes; however, its effects on DNA polymerization speed and accuracy have not been investigated genome-wide. Such an inquiry is critical for understanding neurological diseases and cancer genome instability. Here we present the first simultaneous examination of DNA polymerization kinetics and errors in the human genome sequenced with Single-Molecule-Real-Time technology. We show that polymerization speed differs between non-B and B-DNA: it decelerates at G-quadruplexes and fluctuates periodically at disease-causing tandem repeats. Analyzing polymerization kinetics profiles, we predict and validate experimentally non-B DNA formation for a novel motif. We demonstrate that several non-B motifs affect sequencing errors (e.g., G-quadruplexes increase error rates) and that sequencing errors are positively associated with polymerase slowdown. Finally, we show that highly divergent G4 motifs have pronounced polymerization slowdown and high sequencing error rates, suggesting similar mechanisms for sequencing errors and germline mutations.

Summary Tandem DNA repeats can be sequenced with long-read technologies, but cannot be accurately deciphered due to the lack of computational tools taking high error rates of these technologies into account. Here we introduce Noise-Cancelling Repeat Finder (NCRF) to uncover putative tandem repeats of specified motifs in noisy long reads produced by Pacific Biosciences and Oxford Nanopore sequencers. Using simulations, we validated the use of NCRF to locate tandem repeats with motifs of various lengths and demonstrated its superior performance as compared to two alternative tools. Using real human whole-genome sequencing data, NCRF identified long arrays of the (AATGG)n repeat involved in heat shock stress response. Availability and implementation NCRF is implemented in C, supported by several python scripts, and is available in bioconda and at https://github.com/makovalab-psu/NoiseCancellingRepeatFinder. Supplementary information Supplementary data are available at Bioinformatics online.

DNA sequence motifs and epigenetic modifications contribute to specific binding by a transcription factor, but the extent to which each feature determines occupancy in vivo is poorly understood. We addressed this question in erythroid cells by identifying DNA segments occupied by GATA1 and measuring the level of trimethylation of histone H3 lysine 27 (H3K27me3) and monomethylation of H3 lysine 4 (H3K4me1) along a 66 Mb region of mouse chromosome 7. While 91% of the GATA1-occupied segments contain the consensus binding-site motif WGATAR, only approximately 0.7% of DNA segments with such a motif are occupied. Using a discriminative motif enumeration method, we identified additional motifs predictive of occupancy given the presence of WGATAR. The specific motif variant AGATAA and occurrence of multiple WGATAR motifs are both strong discriminators. Combining motifs to pair a WGATAR motif with a binding site motif for GATA1, EKLF or SP1 improves discriminative power. Epigenetic modifications are also strong determinants, with the factor-bound segments highly enriched for H3K4me1 and depleted of H3K27me3. Combining primary sequence and epigenetic determinants captures 52% of the GATA1-occupied DNA segments and substantially increases the specificity, to one out of seven segments with the required motif combination and epigenetic signals being bound.

The clinical features, age at onset and survival of 152 patients with von Hippel-Lindau disease were studied. Mean age at onset was 26.3 years and 97 per cent of patients had presented by aged 60 years. Retinal angioma was the first manifestation in 65 patients (43 per cent), followed by cerebellar haemangioblastoma (n = 60, 39 per cent) and renal cell carcinoma (n = 15, 10 per cent). Overall, 89 patients (59 per cent) developed a cerebellar haemangioblastoma, 89 (59 per cent) a retinal angioma, 43 (28 per cent) renal cell carcinoma, 20 (13 per cent) spinal haemangioblastoma and 11 (7 per cent) a phaeochromocytoma. Renal, pancreatic and epididymal cysts were frequent findings but their exact incidence was not accurately assessed. Mean age at diagnosis of renal cell carcinoma (44.0 +/- 10.9 years) was significantly older than that for cerebellar haemangioblastoma (29.0 +/- 10.0 years) and retinal angioma (25.4 +/- 12.7 years). The probability of a patient with von Hippel-Lindan disease developing a cerebellar haemangioblastoma, retinal angioma or renal cell carcinoma by age 60 years was 0.84, 0.7 and 0.69, respectively. A comprehensive screening protocol for affected patients and at-risk relatives is presented, based on detailed analysis of age at onset data for each of the major complications. Median actuarial survival was 49 years, with renal cell carcinoma the leading cause of death.

The effects of increased and low dietary sucrose levels on the biochemical activities and composition of 4- and 12-day plaque were investigated in eight young men. The subjects were fed supervised, nutritionally adequate diets. Dextran hydrolase, levan hydrolase, and total carbohydrate content were increased during a sucrose-rich diet while invertase activity was decreased. Base-extractable carbohydrate was decreased in four-day plaque during the sucrose-rich diet. Glucosyltransferase and fructosyltransferase activity, buffer-soluble carbohydrate, and buffer-soluble protein were also determined.

Four batches of potassium acid phosphate crystals of different particle sizes (10–20,20–30,30–40,40–50 mesh) were added at a 1.7% level to a caries-producing diet, thus doubling its phosphorus content. The unsupplemented diet (A) and the four supplemented diets (B, C, D, E, respectively) were fed to groups of twenty-four rats for 98 days, beginning at 8 days of age. The molar teeth were then scored for dental caries development. The average caries scores of groups A to E were 38.8, 36.4, 7.7, 13.5 and 11.8, respectively. Thus, the score of the group (B) fed the coarse 10–20 mesh phosphate supplement was essentially the same as that of the control group. The greatest reduction in caries development (80 per cent) was observed in the group (C) fed the 20–30 mesh phosphate. The two groups (D, E) fed finer granulations of phosphate showed 65 and 70 per cent reductions, respectively. These results indicate that the cariostatic action of potassium acid phosphate is predominantly on the tooth surface, and that the particle size of a dietary supplement of phosphate can significantly influence its cariostatic action.

– We have established a complete system for quantitation and analysis of dental plaque from human subjects fed nutritionally adequate, well characterized diets containing high and low levels of sucrose. Dietary sucrose level did not significantly alter the quantity of plaque produced. High-sucrose diet increased plaque Streptococcus mutans populations. Dextran and levan hydrolase activities were also directly related to dietary sucrose level.

The fatty acid composition of rat molars and incisors was essentially the same whether starch or sucrose was the dietary carbohydrate. A slight increase in palmitic acid and decrease in unsaturated fatty acids was noted in the rats fed starch as compared with those fed sucrose.

Three lots of cold-pressed soybean oil were treated with bubbling oxygen for 70, 80, and 180 hrs. at 70°C. and fed to rats at a level of 18% in diets which were nutritionally adequate but devoid of vitamin A. Untreated soybean oil was fed in similar control diets. Subgroups of 15 weanling rats each were given graded injections of vitamin A acetate intramuscularly each week. Diarrhea developed in the rats fed the diets containing oxidized oil. This condition soon subsided in the groups receiving vitamin A injections but not in the vitamin A-free group. Diarrhea was not noted in the rats receiving the untreated soybean oil, without respect to the amount of vitamin A they received. The rats on the vitamin A-free diets developed deficiency more rapidly when the diet contained oxidized rather than the untreated oil. The food efficiencies of the groups fed the oxidized oils were lower than the controls. The intestines of the groups receiving the oxidized oils were distended with fluid and were hemorrhagic. Enlarged kidneys were noted in the vitamin A-deficient control as well as in test rats. The retroperitoneal lipids of the groups on the oxidized oil were less unsaturated, had lower refractive indices, higher peroxide values, and higher carbonyl values than comparable groups fed the control oil. Vitamin A deficiency decreased the unsaturation of the kidney and liver lipids but increased that of the retroperitoneal lipids. Injections of increasing amounts of vitamin A produced increases in the unsaturation of the body lipids. The kidney lipids of the groups on the oxidized oil diets were less unsaturated and contained more peroxidic compounds than the controls. Vitamin A deficiency increased the peroxidic compounds in the kidney and liver lipids, even in rats fed the control oil. The liver lipids of the groups fed oxidized oil were less unsaturated, lower in vitamin A content, and higher in peroxide compounds than the controls. The vitamin A content of the whole blood varied in relation to the amounts injected. The content of tocopherol in the tissues were not affected significantly by the oxidized oil in the diet. The evidence indicates that severely oxidized oil may destroy vitamin A in the tissue of the rat, thereby hastening the development of deficiency on vitamin A-free diets, reducing the storage of injected vitamin A, and increasing the vitamin A requirement. These effects are with abused oil and should not be interpreted to mean that the mildly oxidized oils and fats, such as those in the diets of human beings in this country are toxic.

Weanling rats were maintained on a cariogenic diet for three or six weeks. Two control groups were given this diet ad libitum; the food intake of two other groups was restricted. The restricted groups gained less weight and developed less dental caries. Phospholipids and free fatty acids were the main lipid fractions in molars and incisors. Food restriction altered the amounts of these two fractions in the teeth.

Caries incidence was generally lower in rats fed a cariogenic diet that contained four different oils and fats at a 9% level than in those fed a diet with a 3% level of the same oil or fat. The amount of linoleic acid in pooled samples of dentin and enamel extracted from the molars and incisors was related to the amount of linoleic acid in the diet.

Because numerous investigators have reported that phosphates are effective in reducing the development of dental caries when fed in the diets of hamsters and rats, it is important to determine whether phosphates are more effective if ingested at certain stages of tooth development and maturation than at other stages. A factorial design was used to study the effect of 1.31 per cent sodium trimetaphosphate mixed into a caries-producing diet and fed to rats during four stages of tooth development (1) during the last 15 days before birth, (2) during the first 20 days after birth or before completion of the eruption of the molar teeth, (3) during the period from 20 to 40 days of age or immediately following eruption of the molar teeth, (4) during 40–80 days of age, or later in the post-eruptive maturation of the molar teeth. All 16 combinations of these four periods were tested. The rats were sacrificed when 80 days old, and the 12 molar teeth in each rat were scored for caries development. Analysis of variance showed that feeding the phosphate during pregnancy had no influence on dental caries and a slight one when fed during the first 20 days. The supplement was highly effective when offered to rats immediately following eruption from 20 to 40 days or from 40 to 80 days of age. The cariostatic effect in rats appears to be due mostly to a local action of the phosphate. Still greater reductions in caries scores were noted when the phosphate was fed during more than one stage of tooth development. Thus, stage of development of the tooth and the length of the feeding period are of critical importance in evaluating the cariostatic action of phosphates in animals and presumably in human beings.

1. 1. Sorbitan monolaurate, polyoxyethylene sorbitan monolaurate, and polyoxyethylene monolaurate were fed at levels of 5 and 15% in nutritionally complete synthetic diets to groups of 36 hamsters for 68 days. 2. 2. All groups fed these test substances showed a poorer weight increase and less efficient utilization of the diet than control groups fed 5 and 15% hydrogenated oils. 3. 3. Diarrhea developed in all groups except those fed 5% sorbitan monolaurate and those fed 5, 7.5 and 15% hydrogenated oils. 4. 4. Compared to the control groups, mortality was higher in all the test groups except that group which received 5% sorbitan monolaurate; the mortality was higher in the groups fed the test substances at the 15% level than at the 5% level. No hamster survived the feeding of 15% polyoxyethylene sorbitan monolaurate or 15% polyoxyethylene monolaurate. 5. 5. The histopathological results indicated that the three test substances were irritating to the gastrointestinal tract and the tubular epithelium of the kidneys and that they inhibited gonadal maturation.

It has been possible to show by this series of tests that the ingestion by the rat of large amounts of Vitamin D in the form of tuna liver oil or tuna liver oil concentrate will cause calcification of certain tissues.

Nutrients are destroyed when foods are processed largely because they are sensitive to the pH of the solvent, to oxygen, light and heat, or combinations of these. Trace elements, especially copper and iron, and enzymes may catalyze these effects.

The effect of food restriction on the quality and quantity of lipids in the teeth of rats was studied. Control animals were fed a nutritionally adequate diet ad libitum while test animals were restricted to 65 per cent as much diet day by day. Both groups were started on the regimen at weaning. Approximately half were sacrificed at 43 days of age. and the remainder at 78 days of age. Compared to control group, the rats fed the restricted diet gained less body weight and retained less lipids in teeth. The tooth lipids of test animals were predominantly phospholipids and cholesterol, while those of control animals were mostly triglycerides and phospholipids. Palmitic, stearic and oleic acids were the major fatty acids in the teeth of rats of both groups. The ratio of oleic acid to total fatty acids in teeth was higher in the test animals than in the control animals, and it increased sharply in the teeth of control animals as they advanced from 43 to 78 days of age. However, the fatty acid composition of tooth lipids of test animals did not change significantly during this period. Thus, food restriction can affect the lipid composition of the teeth of rats. The structural lipids (phospholipids and cholesterol) are retained while the storage lipids (triglycerides) are reduced.

Fifty patients who received a new hypocholesterolemic agent, probucol, in a dosage of 0.5 gm twice daily, were studied for one year. Mean pretreatment serum cholesterol and triglyceride values were 329 mg/100 ml and 360 mg/100 ml respectively. Baseline lipoprotein electrophoreses showed 17 Fredrickson phenotypic patterns of type 2, 6 of type 3, 10 of type 4, 3 of type 5, and 14 non-definitive patterns. After twelve months of treatment with probucol, the mean serum cholesterol level was 263 mg/100 ml (a minus 20 per cent change) and the mean triglyceride level was 293 mg/100 ml (a minus 19 per cent change) for all patients. The median baseline cholesterol level was 311 mg/100 ml and the median cholesterol value during twelve months of therapy was 252 mg/100 ml, a reduction of 19 per cent. The median baseline triglyceride level was 174 mg/100 ml and the median triglyceride value during twelve months of therapy was 147 mg/100 ml, a reduction of 16 per cent. Significant changes were noted in the serum lipoprotein patterns; the majority of type 2 and type 3 changed to the non-definitive pattern, but the majority of type 4 and type 5 remained unchanged. Seven patients had xanthelasmas, but all these lesions decreased in size and 3 lesions disappeared during probucol therapy. Side effects were minimal. About a third of the patients had mild transient flatulence or slight transient eosinophilia. There was no effect on prothrombin time. Probucol appears to be a safe and effective hypolipidemic agent. It can also induce changes in serum lipoprotein patterns and markedly reduce the size of xanthelasmas.

Publisher Summary Research on the influences of storage and processing on the retention of tocopherols in foods of plant and animal origin has been hampered seriously because methods for the estimation of total tocopherol were unsatisfactory and complicated, because the various tocopherols vary in vitamin E activity, and because these several forms of tocopherol here difficult to separate and quantitate individually. To discuss the data on food materials, where the interest is in vitamin E activity rather than in total tocopherol content, it is necessary to use only those scientific reports in which either bioassays were used or the amounts of the individual tocopherols were reported. From the latter data it is possible to calculate the equivalent “alpha-tocopherol activity” just as it has become customary to calculate the various carotenes in plant tissues in terms of “beta-carotene activity” or “vitamin A activity.” It is too soon to use this approach to the tocopherol data, mainly because the analytical procedure was developed only recently and is still being perfected, and because it is not yet agreed what the conversion factors should be. This chapter is a survey that serves to give emphasis to certain of the properties of the tocopherols and will encourage scientists to reinvestigate and extend this information now that more specific and sophisticated methods are becoming available.

Publisher Summary Thiamine hydrochloride crystallizes into colorless, monoclinic needles, which have a melting point of about 250°C, a characteristic smell, and a slightly bitter taste. These crystals are stable to the atmospheric oxygen. They are very soluble in water, much less so in alcohol, and insoluble in ether and other fat solvents. The activity of thiamine is very specific. In several microorganisms, the thiamine is replaced by one or both of its pyrimidine and thiazole moieties. Thiamine is sold commercially in the form of two salts: (1) the chloride hydrochloride and (2) the mononitrate. The hydrochloride is official in most countries, whereas the mononitrate is especially used in the United States. The United States, England, and Switzerland are big producers of thiamine. The first methods used in the estimation of thiamine were animal assays. By using microbiological methods, many samples can be inexpensively assayed in a short time. Presently, the most widely used method for the determination of thiamine, particularly in foodstuff and feeds, is the fluorometric thiochrome method.

Publisher Summary This chapter discusses the isolation, physical and chemical properties, and synthesis of choline. Choline is widely distributed in biological materials as free choline, acetylcholine, and more complex phospholipids and their metabolic intermediates. Choline is characterized by a trimethyl quaternary nitrogen. Choline has been obtained from a variety of tissues and fluids since their original isolations. The earliest methods employed for isolating choline from biological extracts were dependent on the use of various sensitive though nonspecific precipitants. Choline is precipitated from alcoholic solutions as the double salt of platinum, gold, or mercury chlorides. The periodide separation is generally considered one of the most sensitive methods of precipitating choline. The chapter describes the industrial preparation of choline. The reaction between trimethylamine and either ethylene chlorohydrin or ethylene oxide is commonly used in the manufacture of choline. The studies on choline and its derivatives have emphasized the biochemical importance of these compounds as structural components of tissues, as intermediates in vital metabolic reactions, and as specific chemical reactants of marked biological potency.

Lipids and fatty acids were extracted from the teeth of 16 animal species and were characterized by thin-layer and gas-liquid chromatography. The types of lipids and fatty acids in the various teeth were similar, but the amounts were different. The major lipids were glycerides, cholesterol, cholesterol esters, and phospholipids; the major fatty acids were palmitic, stearic, and oleic acids. Trace amounts of many other lipids and fatty acids were present.

Publisher Summary The group of the natural vitamins K comprises a series of naphthoquinone derivatives that contain an unsaturated side chain of four to thirteen isoprene units linked together head to tail. This chapter discusses the chemistry of the naturally occurring K vitamins and their close analogs. The vitamins K are found in plants, microorganisms, and animals. The vitamins K are isolated by fairly simple procedures. The extraction of the natural sources, either directly or after saponification, with suitable solvents followed by chromatographic purification yields the desired products. Chromatographic methods are employed for the isolation, purification, and identification of natural and synthetic isoprenoid naphthoquinones. The most common adsorbents used for column chromatography are aluminum oxide, silicic acid, silica gel, Decalso, Florisil, Permutite, Darco, and magnesium and zinc carbonate. The additional purification, by reversed phase chromatography on polyethylene powder, yields material of excellent purity, as exemplified by the purification of phylloquinone from alfalfa.

1. 1. Sorbitan monolaurate (Span 20), polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene monolaurate (G-2129), and polyoxyethylene monostearate (Myrj 45) were fed to rats at a level of 25% of a synthetic diet in two series of experiments for 59 and 70 days, respectively. 2. 2. Rats fed each of these test substances showed a poorer weight increase and utilized their diets less efficiently than control groups fed hydrogenated oils (either alone, or alone with restricted intake, or with inert Celluflour). 3. 3. The test substances (except polyoxeythylene monostearate) caused a severe diarrhea to develop within a few days which generally persisted throughout the entire test period. 4. 4. The presence of these surface-active agents (except polyoxyethylene monostearate) in the diets increased the mortality in rats, sorbitan monolaurate being the most harmful in this respect. 5. 5. Rats fed sorbitan monolaurate developed a necrosis of the hind legs and tail and became anemic. 6. 6. Rats fed polyoxyethylene sorbitan monolaurate showed upon autopsy distended caeca which were full of liquid and gas. 7. 7. Several rats fed polyoxyethylene monolaurate in the diet showed oxalate stones in the bladder. 8. 8. Feeding of the test substances was associated with histopathological evidences of irritation of the gastrointestinal tract, mild and probably reversible degenerative alteration of the kidney tubules, incomplete spermatogenesis, pneumonitis, and reticuloendothelial hyperplasia in the spleen. Sorbitan monolaurate feeding produced focal hepatic necrosis and fat metamorphosis.

THE aetiology of dental caries has defied complete understanding, despite the fact that the production of bacterial acids at the sites of caries attack has been recognized since the days of Miller, some ninety years ago. Although alternatives to acids have at times been considered, they have never been convincing.

In spite of our highly sensitive chromatographic methods for the detection of organic peroxides, we have been unable to detect significant amounts of these peroxides, in the tissues of rats and chicks fed alpha -tocopherol- deficient or vitamin A-deficient diets, or fed oxidized oils containing peroxides, or subjected to high doses of x radiation. Therefore, it is reasonable to conclude that whatever peroxides are formed in living tissues are too highly unstable to survive separation from these tissues, provided adequate techniques are employed to avoid oxidation during separation. If significant amounts of peroxides are formed in living tissues, we must devise methods for their detection in vivo.