Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer.

Mutational processes underlie cancer initiation and progression. Signatures of these processes in cancer genomes may explain cancer etiology and could hold diagnostic and prognostic value. We developed a strategy that can be used to explore the origin of cancer-associated mutational signatures. We used CRISPR-Cas9 technology to delete key DNA repair genes in human colon organoids, followed by delayed subcloning and whole-genome sequencing. We found that mutation accumulation in organoids deficient in the mismatch repair gene MLH1 is driven by replication errors and accurately models the mutation profiles observed in mismatch repair-deficient colorectal cancers. Application of this strategy to the cancer predisposition gene NTHL1, which encodes a base excision repair protein, revealed a mutational footprint (signature 30) previously observed in a breast cancer cohort. We show that signature 30 can arise from germline NTHL1 mutations

DECREASE AND INCREASE IN RESIDUAL RIDGES AFTER EXTRACTION OF TEETH IN MONKEYS (PART I)

The purpose of this study is to prevent and control the reduction of residual ridges. The subjects used in this study consisted of 19 crab-eating monkeys which were divided into four groups according to the extraction area. These categories are 1U0L group (extracted on M1), 1U5L group (extracted on M1 and M3M2M1P2P1), 0U5L group (extracted on M3M2M1P2P1), and 5UlL group (extracted on M3M2M1P2P1 and M1). Impressions were taken before the extraction. Further impressions were taken at three week, six week, three month, six month, one year, and two year intervals after the extraction in order to observe morphological changes. Casts were made immediately thereafter and the cross-sectional areas of the residual ridges were measured by a standardized method with the aid of a Kubuskraniophor and a diagraph. The measurements taken of the right side, which was operated on, and the left side, which served as the control, were compared in order to observe changes in the form of the edentulous area. The results were as follows: 1) Increase in the Residual Ridge Areas: It is highly significant that the measurements of the M11 section of the 1U5L group and the M1 section of the 5UlL group gradually decreased until the sixth week, and then began to increase until the areas were approximately equivalent to the measurements of the areas before the extraction after a two year period. This may be due to the fact that along with the elongation of the neighboring teeth, the alveolar bone grew to such an extent that the resorption rate was surpassed. 2) Decrease in the Residual Ridges Areas: With the exception of the above-mentioned sections, almost all of other sections responded in the expected manner, that is, there was a sharp decrease in the areas of these sections. The decrease took place rapidly. Seventy to 80% of the total loss occurring over the two-year period took place in the first three months. After a sharp decline in the initial three-month period, the process continued at a slower pace. This gradual decrease after a short period of rapid decrease typifies the standard pattern of the edentulous resorption process

DECREASE AND INCREASE IN RESIDUAL RIDGES AFTER EXTRACTION OF TEETH IN MONKEYS (PART II).

The goal of this study is to reveal (1) the rate of elongation of upper alveolar process after loss of antagonistic teeth, and (2) a change in residual ridge area after extraction of upper elongated teeth. The subjects used in this study were seven crab-eating monkeys, which were divided into two groups; 0U5L group (extracted on M3M2M1P2P1) and 5U5L group (extracted on M3M2M1P2P1 and two years later M3M2M1P2P1). Three monkeys were in each group. In order to observe the morphological changes, impressions were taken before the extraction and at fixed intervals of three weeks, six weeks, three months, six months, one year, two years, and three years (only in the 5U5L group) after the extraction. Casts were made immediately thereafter. Then the changes in the height and cross-sectional area of the alveolar process or the residual ridge were measured. The method was the same as that described in Part I. The results were as follows: 1) 0U5L group: In the first six week period after extraction of antagonistic teeth, the height of the upper alveolar process increased rapidly, and thereafter continued to increase over the two year period. Also the change in the cross sectional area displayed the same tendency as the growth in the height of the alveolar process. 2) 5U5L group: In the first three week period after extraction of the elongated teeth, height of the residual ridge decreased rapidly, and from then on, decreased gradually over the next three year period. However, even after the decrease in height over the three year period, the height of the residual ridge was still greater than the process before elongation of the teeth. The residual ridge area displayed the typical pattern of “rapid decrease followed by a gradual decrease”. However, even after three years the area was still slightly greater than that before the elongation. Thus it is clear that, unlike the decrease in height and area of the residual ridge following extraction of normally occluded teeth, the decrease in the elongated ridge appeared to be difficult

Prenatal organochlorine pesticide exposure and the disruption of steroids and reproductive hormones in cord blood : The Hokkaido study

Certain organochlorine pesticides (OCPs) are designated as persistent organic pollutants and are regulated in many countries. The effects of OCPs on pediatric endocrinology are a concern; however, only limited data exist from human studies on maternal OCP exposure and its effects on infants' hormone levels. This study was conducted as part of the Hokkaido Study Sapporo Cohort, a prospective birth cohort study in Japan. Participants included 514 women who enrolled at 23-35 weeks of gestation between 2002 and 2005; maternal blood samples were collected in late pregnancy, and 29 OCPs were measured. Reproductive and steroid hormone levels in cord blood were also determined. Characteristics of mothers and their infants were obtained from self-administered questionnaires and medical records. Ultimately, 232 samples with both OCP and hormone data were analyzed. Fifteen of 29 investigated OCPs were detected in over 80% of the samples, with p,p'-dichlorodiphenyldichloroethylene showing the highest concentration (median value: 619 pg/g-wet). The association between OCPs and sex hormone levels varied by sex. Linear regression models after sex stratification showed that chlordanes, cis-hexachlorobenzene, heptachlor epoxide, Mirex, and toxaphenes in maternal blood were inversely associated with testosterone, cortisol, cortisone, sex hormone-binding globin, prolactin, and androstenedione-dehydroepiandrosterone (DHEA) and testosterone-androstenediones ratios among boys. Furthermore, these OCPs were positively correlated with DHEA, follicle stimulating hormone (FSH), and adrenal androgen-glucocorticoid and FSH-inhibin B ratios among boys. In categorical quartile models, testosterone and DHEA were inversely and positively associated with OCPs, respectively. Estradiol-testosterone and adrenal androgen-glucocorticoid ratios tended to increase with increasing OCP concentrations in the higher quartile, while the testosterone-androstenedione ratio tended to decrease. Sex hormone-binding globulin and prolactin showed an inverse association with OCPs. Among girls, the linear regression model showed that only p,p'-dichlorodiphenyltrichloroethane was inversely associated with the level of DHEA and the adrenal androgen-glucocorticoid ratio, but was positively associated with cortisone levels. However, no associations were observed using the quartile categorical model. These results suggest that prenatal exposure to OCPs disrupt reproductive hormones of fetuses in utero among boys, even at relatively low levels