Multiplexed genome editing by in vivo electroporation of Cas9 ribonucleoproteins effectively induces endometrial carcinoma in mice

Synergistic effects among multiple gene mutations are involved in cancer development and progression. However, developing genetically modified mouse models to analyze various combinations of mutations is extremely labor-intensive and time-consuming. To address these problems, we developed a novel method for in vivo multiplexed genome editing of the murine uterus to model human endometrial carcinoma (EMC). To do this, we injected a CRISPR-Cas9 ribonucleoprotein complex into the uterine cavity of adult female mice, followed by electroporation. Evaluation of reporter mice demonstrated that genome editing occurred specifically in uterine epithelial cells, which are the origin of EMCs. Simultaneous targeting of Pten/Trp53/Lkb1, or targeting of Pten/Lkb1 along with the Ctnnb1ΔEx3 mutation, resulted in efficient generation of invasive tumors in wild-type females within 3 months. This novel method will enable rapid and easy validation of many combinations of gene mutations that lead to endometrial carcinogenesis.     

Difference in Allelic Expression of Genes Probably Associated with Tumor Progression in Murine Fibrosarcomas and Cell Lines

Allelic expression was examined by single-strand conformation polymorphism analysis in murinc fibrosarcomas from inter-subspecific F₁ mice between C57BL/6 and MSM. Ten genes encoding p53, mdni2, E-cadherin, 72 kD metalloproteinase and its inhibitor (Timp2), thymidine kinasc and four glucose transporters (Gluts) were examined. These genes were chosen because of their probable association with tumor development and progression. In some of the tumors and cell lines, p53, E-cadherin and Glut3 genes showed remarkable differences in allelic expression, one allele being poorly expressed. The allele-specificity persisted in nine cell lines obtained by repeated transplantations from one tumor. These results suggest that expression of some genes is allele-specific in tumor cells and the pattern of specificity is stable. Such a decrease or a loss of expression in one of the alleles may be functionally equivalent to the loss of heterozygosity of the gene, and therefore this may confer malignant properties on tumor cells. It is also suggested that differential expression of two alleles is a common event in tumor cells.     

Comparison of Incorporation and Extension of Nucleotides in vitro opposite 8-Hydroxyguanine (7,8-Dihydro-8-oxoguanine) in Hot Spots of the c-Ha-ras Gene

DNA templates with 8-hydroxyguanine (7,8-dihydro-8-oxoguanine, oh⁸Gua) at a site corresponding to the first or second position of codon 12 of the c-Ha-ras gene were prepared, and the nucleotides inserted opposite the modified base were compared. The Klenow fragment (KF) of Escherichia coli DNA polymerase I inserted C opposite oh⁸Gua at both positions. Taq DNA polymerase incorporated C and A opposite oh⁸Gua, and the ratio of C to A was higher at the first position than at the second position. DNA polymerase α (pol α) inserted A and C at the first position, and A at the second position of codon 12, indicating that the ratio of C to A was higher at the first position. Moreover, we studied the extensions of bases paired with oh⁸Gua by DNA polymerases with or without 3'-5'exonuclease activity. G and T opposite oh⁸Gua were removed, and subsequently C was inserted by KF. We found that an oh⁸Gua:A pair was recognized by the exonuclease activity of the enzyme and that A was partially subsituted by C. On the other hand, pol α extended only C and A opposite oh⁸Gua. No difference was observed with oh⁸Gua at the two positions. These results indicate that the ratio of nucleotides incorporated opposite oh⁸Gua depends on the sequence context, while there is no particular difference in the extension of base pairs involving oh⁸Gua by DNA polymerases.     

Effects of superior colliculus inhibition on visual motion processing in the lateral suprasylvian visual area of the cat

PURPOSE: To clarify whether visual inputs of the tectothalamocortical pathway influence motion processing within the lateral suprasylvian (LS) area of the cat. METHODS: This study was conducted in five cats. Tungsten microelectrodes were used for recording visual evoked potentials. The electrodes were introduced into the LS area. An array of 120 randomly located dots was projected onto the stimulus field (40 degrees x 40 degrees) in front of the animal by a slide projector. The dots were moved rightward and leftward alternatively with interstimulus intervals by a mirror attached to a galvanometer, the movements of which were controlled by a microcomputer. Each motion sequence consisted of an abrupt onset of motion that continued for 100 msec followed by an abrupt offset and a stationary phase of 900 msec; the total duration of each sequence was thus 1000 msec. The velocity of the motion was varied in 12 steps. The onset of motion was used as the trigger for recording evoked potentials. Single or multiple injections (two to three) of muscimol were made, mainly into the rostral superior colliculus (SC). The amplitudes of evoked potentials before and after the muscimol injection were compared. RESULTS: A large negative wave (N1) with the peak latency of 89.80+/-16.39 msec (mean +/- SD, n = 191) was recorded consistently. The amplitude of N1 was not altered by the muscimol injection into the SC when the velocity of motion was 50 deg/sec or less. When the velocity of motion was 75 deg/sec or more, however, the amplitude of N1 was reduced to 62% to 72% of that noted before the muscimol injection. CONCLUSIONS: These findings suggest that the LS area processes the visual motion inputs reaching through the two parallel pathways, the geniculostriate pathway and the tectothalamocortical pathway, when the velocity of visual motion is 75 deg/sec or more.     

Neonatal hemophilia B with intracranial hemorrhage. Case report

It is uncommon for infants with hemophilia to have excessive bleeding during the neonatal period. Even if bleeding occurs, it rarely becomes life-threatening, such as in intracranial hemorrhage (ICH). We here report a case of a 4-day-old boy who had intracranial hemorrhage as the first complication of hemophilia B. Computerized axial tomography (CT scan) and ultrasonography were very useful for early diagnosis. Only a few cases of neonatal hemophilia with intracranial hemorrhage have been reported, but the occurrence of this complication is probably more frequent. We reviewed seven cases (including our case) with intracranial hemorrhage as the first manifestation of neonatal hemophilia. Although these infants showed good prognosis as to survival, permanent residual neurological deficits remained in all of them. It is emphasized that intracranial hemorrhage due to hemophilia may occur in neonates even without a family history. Urgent neuroimaging and coagulation studies are necessary for an early and adequate diagnosis.     

Anterior cruciate ligament reconstruction with a multistrand hamstring graft fixed with a Ligament Tension Screw: a new device that can be used to readjust graft tension after fixation

We developed a new fixation device for reconstruction of the anterior cruciate ligament with a multistrand hamstring graft. This device allows the graft tension to be readjusted after fixation. This device, called a Ligament Tension Screw, consists of three parts, a screw, a washer, and a post. To create the graft, the tendons were formed into a loop. The device was hung from one end of the loop of the graft and a Kennedy-ligament augmentation device (LAD) artificial ligament was hung at the other end. The Kennedy-LAD artificial ligament was used extraarticularly for fixation at the tibia with double staples. Graft tension was increased by turning the screw, which pulls on the post suspending the graft. Fifty-two patients were evaluated after a minimum 2-year follow-up. The evaluation included physical examination, stability measurement with a KT2000 arthrometer, a Cybex muscle strength measurement, a functional test, and scoring of the knee according to the International Knee Documentation Committee (IKDC). Pivot test was negative in 47 patients (91%), but glide occurred in 5 patients (9%). No patient had a "clunk" or markedly poor result on the pivot test. All patients recovered full extension. Seven patients lost full flexion, but the loss of flexion was less than 5° in all seven. The KT 2000 arthrometer measurement with 133 N anterior drawer force indicated that 46 patients (88%) had less than 3-mm side-to-side difference. The mean muscle strength of the quadriceps and hamstrings was 91% and 94%, respectively, compared with the findings in the contralateral limb. The functional test showed a one-leg hop distance equal to 94% of that the opposite side. According to the final IKDC evaluation, 48 patients were graded as normal or nearly normal, and 4 patients as abnormal; none was severely abnormal. This study demonstrated satisfactory results for the reconstruction with a multistrand hamstring graft fixed with the new Ligament Tension Screw. Received: February 15, 2001 / Accepted: July 30, 2001     

Multicentric Castleman's disease associated with inherited epidermolysis bullosa

Multicentric Castleman's disease (MCD) is a rare disorder characterized by fever, polyclonal hypergammaglobulinemia, and generalized lymphadenopathy. It has three histological characteristics: a recognizable architecture, germinal center abnormalities, and plasmacytosis. Inherited epidermolysis bullosa (EB) is also a rare disorder caused by a genetic defect. We report a 43-year-old patient with dystrophic EB, non-Hallopeau-Siemens recessive type or dominant type, displaying clinicopathologic features of MCD. In addition, his serum interleukin-6, which is thought to be responsible for the clinical symptoms in MCD, was elevated.     

Paraovarian cyst with torsion in children

Paraovarian cyst is very uncommon in children, and its preoperative diagnosis is difficult. This report describes 2 cases of surgically proven paraovarian cyst with torsion. It is important to be aware of torsion as a complication of paraovarian cyst. J Pediatr Surg 37:937-940.     

Results of surgical treatment for intrahepatic cholangiocarcinoma and clinicopathological factors influencing survival

BACKGROUND: The results of surgical treatment for intrahepatic cholangiocarcinoma (ICC) and specific factors influencing survival are still unclear. METHODS: Between 1984 and 2001, 62 patients with ICC underwent laparotomy, with a 77 per cent (48 patients) resectability rate. The tumours in these 48 patients were reviewed retrospectively to examine the relationship between gross appearance (mass forming, periductal infiltrating, intraductal growth, and mass forming plus periductal infiltrating) and patient survival, as well as the manner of recurrence. In patients with mass-forming and mass-forming plus periductal infiltrating types, univariate and multivariate analyses of potential prognostic factors were performed. RESULTS: The 1-, 3- and 5-year survival rates were 62, 38 and 23 per cent respectively. All patients with the intraductal growth type remained alive after intervals ranging from 8 to 72 months. Univariate analysis showed multiple hepatic lesions, liver capsule invasion, presence of cancer cells in the resection margin, and high serum carbohydrate antigen (CA) 19-9 level to be significant negative prognostic factors. Lymph node involvement, however, was not identified as a significant prognostic factor. With multivariate analysis, multiple hepatic lesions and high serum CA19-9 concentration were found to be significantly related to prognosis. The most frequent recurrence site was the remnant liver. CONCLUSION: These results suggest that the intraductal growth type of tumour should be treated as a distinct entity compared with other types of ICC. Multiple tumours and high serum CA19-9 level were signs of dismal prognosis, whereas not all patients with lymph node involvement had a poor prognosis.