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Background: Interstitial cystitis (IC)/bladder pain syndrome (BPS) is an unpleasant sensation related to the bladder with lower urinary tract symptoms lasting more than 6 weeks, unrelated to an otherwise identifiable cause. The etiology is likely multifactorial including urothelial abnormalities, neurogenic pain upregulation, and potentially bladder and vaginal microbiome alterations. Despite treatment effectiveness of both bladder instillations and intradetrusor onabotulinumtoxinA injection for this condition, a head-to-head comparison has not been performed., Objective: To compare the efficacy of bladder instillations and intradetrusor onabotulinumtoxinA injection for treatment of IC/BPS., Study Design: Patients with O'Leary-Sant (OLS) questionnaire scores of ≥6, meeting clinical criteria for IC/BPS, and desiring procedural management were randomized to bladder instillations or intradetrusor onabotulinumtoxinA injection. The primary outcome was the difference in OLS scores at 2 months posttreatment between groups. Secondary outcomes included evaluation of sexual function, physical/mental health status, pain, patient satisfaction, treatment perception, retreatment, and adverse event rates., Results: Forty-seven patients were analyzed with 22 randomized to bladder instillations and 25 to onabotulinumtoxinA injection. There were no differences in demographic and clinical characteristics between groups. From baseline to 2 months posttreatment, there was a decrease in OLS subscales in all patients (Interstitial Cystitis Symptom Index [ICSI] -6.3 (confidence interval [CI] -8.54, -3.95), P<.0001; Interstitial Cystitis Problem Index [ICPI] -5.9 (CI -8.18, -3.57), P<.0001). At 2 months posttreatment, patients in the onabotulinumtoxinA group had significantly lower OLS scores compared to those in the bladder instillation group (ICSI 6.3±4.5 [onabotulinumtoxinA] vs 9.6±4.2 [instillation], P=.008; ICPI 5.9±5.1 [onabotulinumtoxinA] vs 8.3±4.0 [instillation], P=.048). The difference in OLS scores between groups did not persist at 6 to 9 months posttreatment. There were no statistically significant differences between baseline and posttreatment time points for the remaining questionnaires. Eight percent of patients who received onabotulinumtoxinA injection experienced urinary retention requiring self-catheterization. Patients who underwent onabotulinumtoxinA injection were significantly less likely to receive retreatment within 6 to 9 months compared to patients who received bladder instillations (relative risk 13.6; 95% CI, 1.92-96.6; P=.0002). There were no differences between groups regarding patient satisfaction, perception of treatment convenience, or willingness to undergo retreatment., Conclusion: Both onabotulinumtoxinA injection and bladder instillations are safe, effective treatments for patients with IC/BPS, with significant clinical improvement demonstrated at 2 months posttreatment. Our findings suggest that intradetrusor onabotulinumtoxinA injection is a more effective procedural treatment for this condition than bladder instillation therapy and associated with decreased rates of retreatment., (Published by Elsevier Inc.)

The ability to quickly perceive others' rank minimizes costs by helping individuals behave appropriately when interacting with strangers. Indeed, humans and at least some other species can quickly determine strangers' rank or dominance based only on physical features without observing others' interactions or behavior. Nonhuman primates can determine strangers' ranks by observing their interactions, and some evidence suggests that at least some cues to dominance, such as facial width-to-height ratio (fWHR), are also present in other primates. However, it is unknown whether they can determine strangers' rank simply by looking at their faces, rather than observing their interactions. If so, this would suggest selective pressure across the primates on both cues to dominance and the ability to detect those cues accurately. To address this, we examined the ability of male and female tufted capuchin monkeys (Sapajus [Cebus] apella) to categorize images of the faces of unknown conspecifics (Sapajus from different colonies) and humans (computer-generated and real) as dominant or nondominant based only on still images. Capuchins' categorization of unknown conspecific faces was consistent with fWHR, a cue to dominance, although there was a strong tendency to categorize strangers as dominant, particularly for males. This was true despite the continued correct categorization of known individuals. In addition, capuchins did not categorize human strangers in accordance with external pre-ratings of dominance by independent human raters, despite the availability of the same cue, fWHR. We consider these results in the context of capuchin socio-ecology and what they mean for the evolution of rapid decision-making in social contexts., (© 2024 Wiley Periodicals LLC.)

Prior research regarding the influence of face structure on character judgments and first impressions reveals that bias for certain face-types is ubiquitous, but these studies primarily used decontextualized White faces for stimuli. Given the disadvantages Black men face in the legal system, this study aimed to investigate whether the criminal face-type presented in the context of crime influenced different legal system-type judgments as a function of perpetrator race. In a mixed-model design, participants saw Black and White computer-generated faces that varied in criminality presented with either violent or nonviolent crime scenarios. At test, participants attempted to identify the original perpetrator from a photo array, along with providing penalty severity judgments for the crime committed. Results indicate that when crimes were violent, participants meted harsher penalties overall to Black faces or to high-criminality faces identified as the perpetrator. Furthermore, for violent crimes, participants were more likely to select a face from the photo array that was higher/equally as high in criminality rating relative to the actual perpetrator when memory failed or when the perpetrator was Black. Overall, the findings suggest that when people are making judgments that could influence another's livelihood, they may rely heavily on facial cues to criminality and the nature of the crime; and this is especially the case for Black faces presented in the context of violent crime. The pattern of results provides further support for the pervasive stereotype of Black men as criminal, even in our racially diverse sample wherein 36% identified as Black., (© 2023. The Psychonomic Society, Inc.)

Background: Intradetrusor onabotulinumtoxinA injection is an effective advanced treatment for overactive bladder. While the effective dosages have been well studied, very little data exist on treatment efficacy using differing injection techniques. The objective of this study was to determine whether the efficacy of a reduced injection technique of 5 injection sites was noninferior to the standard technique of 20 injection sites of intradetrusor onabotulinumtoxinA for treatment of overactive bladder., Methods: In this randomized noninferiority trial, men and women seeking treatment of overactive bladder with intradetrusor onabotulinumtoxinA injections were randomized to receive 100 units administered via either a reduced injection technique of 5 injection sites or a standard injection technique of 20 injection sites. Subjects completed a series of standardized questionnaires at baseline and at 4-12 weeks postprocedure to determine symptom severity and treatment efficacy. The primary outcome was treatment efficacy as determined by Overactive Bladder Questionnaire-Short Form with a noninferiority margin of 15 points. Secondary outcomes were incidence of urinary tract infection and urinary retention requiring catheterization., Results: Data from 77 subjects were available for analysis with 39 in the control arm (20 injections) and 38 in the study arm (5 injections). There was a significant improvement in both arms from baseline to follow-up in Overactive Bladder Questionnaire-Short Form and International Consultation on Incontinence Questionnaire scores (p < 0.001). Overall treatment success was 68% with no statistically significant difference between arms. A significant difference between arms was found on the Overactive Bladder Questionnaire-Short Form quality of life survey favoring the control arm (confidence interval [CI]: 0.36-20.5, p = 0.04). However, there were no significant differences between arms in the remaining validated questionnaires. The study arm did not demonstrate noninferiority to the control arm. Subjects in the study arm were significantly more likely to express a willingness to undergo the procedure again (odds ratio = 3.8, 95% CI: 1.42-10.67, p = 0.004). Adverse events did not differ between arms., Conclusions: A reduced injection technique for administration of intradetrusor onabotulinumtoxinA demonstrates similar efficacy to the standard injection technique but did not demonstrate noninferiority. Subjects preferred the reduced injection technique over the standard technique. A reduced injection technique is a safe and effective alternative to the standard technique., (© 2022 The Authors. Neurourology and Urodynamics published by Wiley Periodicals LLC.)

Objectives: This study aimed to evaluate transient urinary retention in women undergoing outpatient midurethral sling placement who received preoperative gabapentin (treated) versus those who did not (untreated). Secondary outcomes included unexpected admission rates, analgesic usage, time to discharge, and pain., Methods: This was a retrospective cohort study including women who underwent outpatient midurethral sling placement from 2015 to 2019. Exclusion criteria included suprapubic catheter placement, planned overnight admission, abnormal preoperative postvoid residual volume, and prolonged postoperative catheterization. Logistic regression was performed to evaluate gabapentin usage and urinary retention after adjusting for patient characteristics., Results: Three hundred two women met the inclusion criteria, with 19.5% experiencing urinary retention after midurethral sling placement. Women older than 65 years were more likely to have urinary retention than those aged 18-65 years (29.8% vs 17.6%, P = 0.054). Of treated participants, 26% had urinary retention versus 18% of untreated participants (P = 0.162). Adjusting for age, parity, pain, operative time, blood loss, sling type, analgesic, scopolamine patch, or hemostatic agent use, treated participants had 72% higher odds of urinary retention (adjusted odds ratio, 1.72; 95% confidence interval, 0.88-3.38; P = 0.113). There was no difference in unexpected admission, analgesic usage, time to discharge, or pain between groups., Conclusions: One of 5 women had urinary retention after outpatient midurethral sling placement. Although no statistically significant difference was found in urinary retention between groups, the odds of urinary retention in the treated group were increased. Because there was no difference in pain, analgesic usage, or time to discharge between groups, investigation regarding gabapentin use for outpatient urogynecologic surgery is needed., Competing Interests: The authors have declared they have no conflict of interest., (Copyright © 2021 American Urogynecologic Society. All rights reserved.)

Background: Pelvic reconstructive surgery may cause significant postoperative pain, especially with posterior colporrhaphy, contributing to a longer hospital stay and increased pain medication utilization. Regional blocks are being increasingly utilized in gynecologic surgery to improve postoperative pain and decrease opioid usage, yet preoperative pudendal blocks have not been used routinely during posterior colporrhaphy., Objective: This study aimed to compare the effect of preoperative regional pudendal nerve block using a combination of 1.3% liposomal and 0.25% plain bupivacaine vs 0.25% plain bupivacaine alone on vaginal pain after posterior colporrhaphy on postoperative days 1, 2, and 3. We hypothesized that there would be a reduction in vaginal pain scores for the study group vs the control group over the first 72 hours., Study Design: This was a double-blinded, randomized controlled trial that included patients undergoing a posterior colporrhaphy, either independently or in conjunction with other vaginal or abdominal reconstructive procedures. Patients were block randomized to receive 20 mL of either a combination of 1.3% liposomal and 0.25% plain bupivacaine (study) or 20 mL of 0.25% plain bupivacaine (control) in a regional pudendal block before the start of surgery. Double blinding was achieved by covering four 5-mL syringes containing the randomized local anesthetic. After induction of anesthesia, a pudendal nerve block was performed per standard technique (5 mL superiorly and 5 mL inferiorly each ischial spine) using a pudendal kit. The primary outcome was to evaluate postoperative vaginal pain using a visual analog scale on postoperative days 1, 2, and 3. Secondary outcomes included total analgesic medication usage through postoperative day 3, postoperative voiding and defecatory dysfunction, and impact of vaginal pain on quality of life factors., Results: A total of 120 patients were enrolled (60 in each group). There were no significant differences in demographic data, including baseline vaginal pain (P=.88). Postoperative vaginal pain scores were significantly lower in the combined liposomal and bupivacaine group at all time points vs the plain bupivacaine group. Median pain scores for the study and control groups, respectively, were 0 (0-2) and 2 (0-4) for postoperative day 1 (P=.03), 2 (1-4) and 3 (2-5) for postoperative day 2 (P=.05), and 2 (1-4) and 3 (2-5) for postoperative day 3 (P=.02). Vaginal pain scores increased from postoperative day 1 to postoperative days 2 and 3 in both groups. There was a significant decrease in ibuprofen (P=.01) and acetaminophen (P=.03) usage in the study group; however, there was no difference between groups in total opioid consumption through postoperative day 3 (P=.82). There was no difference in successful voiding trials (study 72%, control 82%, P=.30), return of bowel function (P>.99), or quality of life factors (sleep, stress, mood, and activity)., Conclusion: Preoperative regional pudendal block with a combination of liposomal and plain bupivacaine provided more effective vaginal pain control than plain bupivacaine alone for reconstructive surgery that included posterior colporrhaphy. Given the statistically significant decrease in vaginal pain in the study group, this block may be considered as a potential adjunct for multimodal pain reduction in this patient population., (Published by Elsevier Inc.)

Prior research indicates that stereotypical Black faces (e.g., wide nose, full lips) are perceived negatively relative to non-stereotypical faces (face-type bias). The current study investigated whether stereotypical faces may bias the interpretation of a neutral facial expression to seem threatening. Moreover, could biased responses be trained away with feedback? In two experiments, stimuli (face images) were presented in a speeded identification task that included corrective feedback, and participants indicated whether the face stimuli were stereotypical or not and threatening or not. Stimuli were pre-rated by face-type (stereotypical, non-stereotypical) and expression (neutral, threatening). Computational modeling based on General Recognition Theory indicated that training increased perceptual discriminability between all the faces. By the end of training (in both experiments), discriminability for emotional expression was slightly higher for stereotypical faces. Model parameters (for both experiments) also showed that, early in training, decision boundaries were more biased toward the threatening response for stereotypical faces relative to non-stereotypical faces. The results suggest that decision bias may be malleable with training., (© 2020. Springer-Verlag GmbH Germany, part of Springer Nature.)

Faces judged as stereotypically Black are perceived negatively relative to less stereotypical faces. In this experiment, artificial faces were constructed to examine the effects of nose width, lip fullness, and skin reflectance, as well as to study the relations among perceived dominance, threat, and Black stereotypicality. Using a multilevel structural equation model to isolate contributions of the facial features and the participant demographics, results showed that stereotypicality was related to wide nose, darker reflectance, and to a lesser extent full lips; threat was associated with wide nose, thin lips, and low reflectance; dominance was mainly related to nose width. Facial features explained variance among faces, suggesting that face-type bias in this sample was related to specific face features rather than particular characteristics of the participant. People's perceptions of relations across these traits may underpin some of the sociocultural disparities in treatment of certain individuals by the legal system., (© 2021. The Author(s).)

Objective: To demonstrate a unique presentation and surgical treatment of a posterior uteroperitoneal fistula with excision of the fistula tract and repair in multiple layers., Design: Illustrative video presentation. A case report is used to describe potential causes of an uteroperitoneal fistula found in a patient during evaluation of secondary infertility and to highlight surgical technique and management using the principles of fistula repair through a minimally invasive approach. The Institutional Review Board reviewed this video article and it was deemed "not human subject research.", Setting: Tertiary medical center., Patient(s): A 33-year-old G1P1001 woman with a history of a cesarean section presented with secondary infertility, pelvic pain, and dysmenorrhea and was found to have a posterior uteroperitoneal fistula at the time of hysterosalpingography., Intervention(s): The patient underwent an uncomplicated robot-assisted laparoscopic excision of a posterior uteroperitoneal fistula with the use of careful dissection of the fistula tract, continuous reassessment, and tension-free closure in layers., Main Outcome Measure(s): Preoperative diagnosis and surgical management displaying intraoperative techniques for robot-assisted excision of fistula tract and repair of defect., Result(s): The patient underwent robot-assisted operative laparoscopy that revealed a 4-cm mass on the right posterior aspect of the uterus independent from her adnexa. Concomitant hysteroscopy revealed normal endometrium without an evident fistula. During chromopertubation, extravasation was seen into the peritoneal cavity from this mass. The mass and fistula tract were excised without a connection found from her cesarean scar, and reconstruction was performed in multiple layers. Endometriotic lesions were noted intraperitoneally in locations distant from the mass. The patient had significant improvement in her symptoms after surgery., Conclusion(s): We present a unique case of a suspected spontaneous posterior uteroperitoneal fistula in the presence of endometriosis without evidence of a connection to her prior hysterotomy scar. Possible etiologies include an undiagnosed, unrepaired hysterotomy extension or a result of chronic inflammation from deep infiltrating endometriosis. Adverse effects on fertility from uteroperitoneal fistulas may be due to disruption of sperm function or endometrial quality secondary to presence of old blood products sequestered in the fistula or due to resultant inflammation from the same. As in this video case, successful treatment of symptoms resulting from an uteroperitoneal fistula requires removal of the fistula tract. The constellation of pelvic pain, dysmenorrhea, postmenstrual bleeding, and infertility should raise suspicion for an uteroperitoneal fistula., (Published by Elsevier Inc.)

Since it is extremely difficult to establish an animal model for human chromosomal abnormalities, induced pluripotent stem cells (iPSCs) provide a powerful alternative to study underlying mechanisms of these disorders and identify potential therapeutic interventions. In this study we established iPSCs from a young girl with a hemizygous deletion of Xq27.3-q28 who exhibited global developmental delay and intellectual disability from early in infancy. The deletion site on the X chromosome includes Fragile X Mental Retardation 1 (FMR1), the gene responsible for fragile X syndrome, which likely contributes to the patient's neurodevelopmental abnormalities. The FMR1 gene was expressed in approximately half of the iPSC clones we generated while it was absent in the other half due to the random inactivation of normal and abnormal X chromosomes. The normal or absent expression pattern of the FMR1 gene was not altered when the iPSCs were differentiated into neural progenitor cells (NPCs). Moreover, chromosome reactivating reagents such as 5-aza-2-deoxycytidine, trichostatin A, and UNC0638, were tested in an attempt to reactivate the suppressed FMR1 gene in affected iPSC-NPCs. The affected and control isogenic iPSCs developed in this study are ideal models with which to identify downstream consequences caused by the Xq27.3-q28 deletion and also to provide tools for high-throughput screening to identify compounds potentially improving the well-being of this patient population.

Background: Clitoral epidermal inclusions cysts are most frequently seen following trauma, especially female genital mutilation. Spontaneous clitoral epidermal inclusion cysts are rare with an unclear etiology and their impact on later sexual function has not been described., Case: A 15-year-old spontaneously developed a clitoral mass that progressively enlarged over seven years, ultimately leading to secondary anorgasmia. Surgical removal resulted in restoration of normal anatomy and complete return of clitoral function. Final pathology revealed the mass to be an epidermal inclusion cyst., Summary and Conclusion: Clitoral epidermal inclusion cysts typically present in childhood or early adolescence and can lead to sexual dysfunction if left untreated. Physicians must consider the potential sequelae of these cysts when counseling and managing these patients., (Published by Elsevier Inc.)

Background: Relapsing disease is a major challenge after hematopoietic cell transplantation for hematological malignancies. Myxoma virus (MYXV) is an oncolytic virus that can target and eliminate contaminating cancer cells from auto-transplant grafts. The aims of this study were to examine the impact of MYXV on normal hematopoietic stem and progenitor cells and define the optimal treatment conditions for ex vivo virotherapy., Methods: Bone marrow (BM) and mobilized peripheral blood stem cells (mPBSCs) from patients with hematologic malignancies were treated with MYXV at various time, temperature and incubation media conditions. Treated BM cells from healthy normal donors were evaluated using flow cytometry for MYXV infection, long-term culture-initiating cell (LTC-IC) assay and colony-forming cell (CFC) assay., Results: MYXV initiated infection in up to 45% of antigen-presenting monocytes, B cells and natural killer cells; however, these infections were uniformly aborted in >95% of all cells. Fresh graft sources showed higher levels of MYXV infection initiation than cryopreserved specimens, but in all cases less than 10% of CD34(+) cells could be infected after ex vivo MYXV treatment. MYXV did not impair LTC-IC colony numbers compared with mock treatment. CFC colony types and numbers were also not impaired by MYXV treatment. MYXV incubation time, temperature or culture media did not significantly change the percentage of infected cells, LTC-IC colony formation or CFC colony formation., Conclusions: Human hematopoietic cells are non-permissive for MYXV. Human hematopoietic stem and progenitor cells were not infected and thus unaffected by MYXV ex vivo treatment., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain hematologic malignancies, but the risk of graft-versus-host disease (GVHD) is a major limitation for wider application. Ideally, strategies to improve allo-HCT would involve suppression of T lymphocytes that drive GVHD while sparing those that mediate graft-versus-malignancy (GVM). Recently, using a xenograft model, we serendipitously discovered that myxoma virus (MYXV) prevented GVHD while permitting GVM. In this study, we show that MYXV binds to resting, primary human T lymphocytes but will only proceed into active virus infection after the T cells receive activation signals. MYXV-infected T lymphocytes exhibited impaired proliferation after activation with reduced expression of interferon-γ, interleukin-2 (IL-2), and soluble IL-2Rα, but did not affect expression of IL-4 and IL-10. MYXV suppressed T-cell proliferation in 2 patterns (full vs partial) depending on the donor. In terms of GVM, we show that MYXV-infected activated human T lymphocytes effectively deliver live oncolytic virus to human multiple myeloma cells, thus augmenting GVM by transfer of active oncolytic virus to residual cancer cells. Given this dual capacity of reducing GVHD plus increasing the antineoplastic effectiveness of GVM, ex vivo virotherapy with MYXV may be a promising clinical adjunct to allo-HCT regimens., (© 2015 by The American Society of Hematology.)

Rationale: Bone marrow (BM) cell therapy for ischemic heart disease (IHD) has shown mixed results. Before the full potency of BM cell therapy can be realized, it is essential to understand the BM niche after acute myocardial infarction (AMI)., Objective: To study the BM composition in patients with IHD and severe left ventricular (LV) dysfunction., Methods and Results: BM from 280 patients with IHD and LV dysfunction were analyzed for cell subsets by flow cytometry and colony assays. BM CD34(+) cell percentage was decreased 7 days after AMI (mean of 1.9% versus 2.3%-2.7% in other cohorts; P<0.05). BM-derived endothelial colonies were significantly decreased (P<0.05). Increased BM CD11b(+) cells associated with worse LV ejection fraction (LVEF) after AMI (P<0.05). Increased BM CD34(+) percentage associated with greater improvement in LVEF (+9.9% versus +2.3%; P=0.03, for patients with AMI and +6.6% versus -0.02%; P=0.021 for patients with chronic IHD). In addition, decreased BM CD34(+) percentage in patients with chronic IHD correlated with decrement in LVEF (-2.9% versus +0.7%; P=0.0355)., Conclusions: In this study, we show a heterogeneous mixture of BM cell subsets, decreased endothelial colony capacity, a CD34+ cell nadir 7 days after AMI, a negative correlation between CD11b percentage and postinfarct LVEF, and positive correlation of CD34 percentage with change in LVEF after cell therapy. These results serve as a possible basis for the small clinical improvement seen in autologous BM cell therapy trials and support selection of potent cell subsets and reversal of comorbid BM impairment., Clinical Trial Registrations Url: http://www.clinicaltrials.gov. Unique identifiers: NCT00684021, NCT00684060, and NCT00824005., (© 2014 American Heart Association, Inc.)

Acute myelogenous leukemias (AMLs) and endothelial cells depend on each other for survival and proliferation. Monotherapy antivascular strategies such as targeting vascular endothelial growth factor (VEGF) has limited efficacy in treating AML. Thus, in search of a multitarget antivascular treatment strategy for AML, we tested a novel vascular disrupting agent, OXi4503, alone and in combination with the anti-VEGF antibody, bevacizumab. Using xenotransplant animal models, OXi4503 treatment of human AML chloromas led to vascular disruption in leukemia cores that displayed increased leukemia cell apoptosis. However, viable rims of leukemia cells remained and were richly vascular with increased VEGF-A expression. To target this peripheral reactive angiogenesis, bevacizumab was combined with OXi4503 and abrogated viable vascular rims, thereby leading to enhanced leukemia regression. In a systemic model of primary human AML, OXi4503 regressed leukemia engraftment alone and in combination with bevacizumab. Differences in blood vessel density alone could not account for the observed regression, suggesting that OXi4503 also exhibited direct cytotoxic effects on leukemia cells. In vitro analyses confirmed this targeted effect, which was mediated by the production of reactive oxygen species and resulted in apoptosis. Together, these data show that OXi4503 alone is capable of regressing AML by a multitargeted mechanism and that the addition of bevacizumab mitigates reactive angiogenesis.

The deoxycytidine analog 5-aza-2'-deoxycitidine (5-aza-dC) is a potent chemotherapeutic agent effective against selective types of cancer. The molecular mechanism by which 5-aza-dC induces cancer cell death, however, is not fully understood. It has been accepted that the mechanism of toxicity is due to the covalent binding between the DNA methyltransferase (Dnmt) and 5-aza-dC-substituted DNA. In order to define which member of the Dnmt family plays a dominant role in the cytotoxicity, we examined the effect of 5-aza-dC on cell growth and apoptosis in various Dnmt null mutant embryonic stem (ES) cells. Of interest, Dnmt3a-Dnmt3b double null ES cells were highly resistant to 5-aza-dC when compared to wild type, Dnmt3a null, Dnmt3b null, or Dnmt1 null ES cells. The cellular sensitivity to 5-aza-dC correlated well with the expression status of Dnmt3 in both undifferentiated and differentiated ES cells. When exogenous Dnmt3a or Dnmt3b was expressed in double null ES cells, the sensitivity to 5-aza-dC was partially restored. These results suggest that the cytotoxic effect of 5-aza-dC may be mediated primarily through Dnmt3a and Dnmt3b de novo DNA methyltransferases. Further, the ability to form Dnmt-DNA adducts was similar in Dnmt1 and Dnmt3, and the expression level of Dnmt3 was not higher than that of Dnmt1 in ES cells. Therefore, Dnmt3-DNA adducts may be more effective for inducing apoptosis than Dnmt1-DNA adducts. These results imply a therapeutic potential of 5-aza-dC to cancers expressing Dnmt3.

We investigated the potential of mouse embryonic stem (ES) cells to differentiate into hepatocytes in vitro. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, transthyretin, alpha 1-anti-trypsin and albumin, when cultured without additional growth factors and late differential markers of hepatic development, such as tyrosine aminotransferase (TAT) and glucose-6-phosphatase (G6P), when cultured in the presence of growth factors critical for late embryonic liver development. Further, induction of TAT and G6P expression was induced regardless of expression of the functional SEK1 gene, which is thought to provide a survival signal for hepatocytes during an early stage of liver morphogenesis. The data indicate that the in vitro ES differentiation system has a potential to generate mature hepatocytes. The system has also been found useful in analyzing the role of growth factors and intracellular signaling molecules in hepatic development.