FOXP1 orchestrates neurogenesis in human cortical basal radial glial cells.

During cortical development, human basal radial glial cells (bRGCs) are highly capable of sustained self-renewal and neurogenesis. Selective pressures on this cell type may have contributed to the evolution of the human neocortex, leading to an increase in cortical size. bRGCs have enriched expression for Forkhead Box P1 (FOXP1), a transcription factor implicated in neurodevelopmental disorders (NDDs) such as autism spectrum disorder. However, the cell type-specific roles of FOXP1 in bRGCs during cortical development remain unexplored. Here, we examine the requirement for FOXP1 gene expression regulation underlying the production of bRGCs using human brain organoids. We examine a developmental time point when FOXP1 expression is highest in the cortical progenitors, and the bRGCs, in particular, begin to actively produce neurons. With the loss of FOXP1, we show a reduction in the number of bRGCs, as well as reduced proliferation and differentiation of the remaining bRGCs, all of which lead to reduced numbers of excitatory cortical neurons over time. Using single-nuclei RNA sequencing and cell trajectory analysis, we uncover a role for FOXP1 in directing cortical progenitor proliferation and differentiation by regulating key signaling pathways related to neurogenesis and NDDs. Together, these results demonstrate that FOXP1 regulates human-specific features in early cortical development.

Corticogenesis across species at single-cell resolution.

The neocortex (or pallium) consists of diverse cell types that are organized in a highly species-specific manner under strict spatiotemporal control during development. Many of the cell types are present transiently throughout development but contribute to permanent species-specific cortical features that are acquired through evolution. Therefore, capturing cell type-specific biological information has always been an important quest in the field of neurodevelopment. The progress in achieving fine cellular resolution has been slow due to technical challenges. However, with recent advancements in single-cell and multi-omics technologies, many laboratories have begun to successfully interrogate cellular and molecular mechanisms driving corticogenesis at single-cell resolution. In this review, we provide summarized results from many primary publications and several in-depth review articles that utilize or address single-cell genomics techniques to understand important topics, such as cellular and molecular mechanisms governing cortical progenitor proliferation, cell lineage progression, neuronal specification, and arealization, across multiple gyrencephalic (i.e., human and non-human primates) and lissencephalic species (i.e., mouse, reptiles, and songbirds). We also examine findings from recent studies involving epigenomic and posttranscriptional regulation of corticogenesis. In the discussion section, we provide our insights on the challenges the field currently faces as well as promising future applications of single cell technologies.

Photoactivatable Cre recombinase 3.0 for in vivo mouse applications.

Optogenetic genome engineering tools enable spatiotemporal control of gene expression and provide new insight into biological function. Here, we report the new version of genetically encoded photoactivatable (PA) Cre recombinase, PA-Cre 3.0. To improve PA-Cre technology, we compare light-dimerization tools and optimize for mammalian expression using a CAG promoter, Magnets, and 2A self-cleaving peptide. To prevent background recombination caused by the high sequence similarity in the dimerization domains, we modify the codons for mouse gene targeting and viral production. Overall, these modifications significantly reduce dark leak activity and improve blue-light induction developing our new version, PA-Cre 3.0. As a resource, we have generated and validated AAV-PA-Cre 3.0 as well as two mouse lines that can conditionally express PA-Cre 3.0. Together these new tools will facilitate further biological and biomedical research.

Optogenetic Control of Endoplasmic Reticulum-Mitochondria Tethering.

The organelle interface emerges as a dynamic platform for a variety of biological responses. However, their study has been limited by the lack of tools to manipulate their occurrence in live cells spatiotemporally. Here, we report the development of a genetically encoded light-inducible tethering (LIT) system allowing the induction of contacts between endoplasmic reticulum (ER) and mitochondria, taking advantage of a pair of light-dependent heterodimerization called an iLID system. We demonstrate that the iLID-based LIT approach enables control of ER-mitochondria tethering with high spatiotemporal precision in various cell types including primary neurons, which will facilitate the functional study of ER-mitochondrial contacts.

Optimized light-inducible transcription in mammalian cells using Flavin Kelch-repeat F-box1/GIGANTEA and CRY2/CIB1.

Light-inducible systems allow spatiotemporal control of a variety of biological activities. Here, we report newly optimized optogenetic tools to induce transcription with light in mammalian cells, using the Arabidopsis photoreceptor Flavin Kelch-repeat F-box 1 (FKF1) and its binding partner GIGANTEA (GI) as well as CRY2/CIB1. By combining the mutagenesis of FKF1 with the optimization of a split FKF1/GI dimerized Gal4-VP16 transcriptional system, we identified constructs enabling significantly improved light-triggered transcriptional induction. In addition, we have improved the CRY2/CIB1-based light-inducible transcription with split construct optimization. The improvements regarding the FKF1/GI- and CRY2/CIB1-based systems will be widely applicable for the light-dependent control of transcription in mammalian cells.

Inhibition of CDK5 Alleviates the Cardiac Phenotypes in Timothy Syndrome.

L-type calcium channel CaV1.2 plays an essential role in cardiac function. The gain-of-function mutations in CaV1.2 have been reported to be associated with Timothy syndrome, a disease characterized by QT prolongation and syndactyly. Previously we demonstrated that roscovitine, a cyclin-dependent kinase (CDK) inhibitor, could rescue the phenotypes in induced pluripotent stem cell-derived cardiomyocytes from Timothy syndrome patients. However, exactly how roscovitine rescued the phenotypes remained unclear. Here we report a mechanism potentially underlying the therapeutic effects of roscovitine on Timothy syndrome cardiomyocytes. Our results using roscovitine analogs and CDK inhibitors and constructs demonstrated that roscovitine exhibits its therapeutic effects in part by inhibiting CDK5. The outcomes of this study allowed us to identify a molecular mechanism whereby CaV1.2 channels are regulated by CDK5. This study provides insights into the regulation of cardiac calcium channels and the development of future therapeutics for Timothy syndrome patients.

Dual optical recordings for action potentials and calcium handling in induced pluripotent stem cell models of cardiac arrhythmias using genetically encoded fluorescent indicators.

Reprogramming of human somatic cells to pluripotency has been used to investigate disease mechanisms and to identify potential therapeutics. However, the methods used for reprogramming, in vitro differentiation, and phenotyping are still complicated, expensive, and time-consuming. To address the limitations, we first optimized a protocol for reprogramming of human fibroblasts and keratinocytes into pluripotency using single lipofection and the episomal vectors in a 24-well plate format. This method allowed us to generate multiple lines of integration-free and feeder-free induced pluripotent stem cells (iPSCs) from seven patients with cardiac diseases and three controls. Second, we differentiated human iPSCs derived from patients with Timothy syndrome into cardiomyocytes using a monolayer differentiation method. We found that Timothy syndrome cardiomyocytes showed slower, irregular contractions and abnormal calcium handling compared with the controls. The results are consistent with previous reports using a retroviral method for reprogramming and an embryoid body-based method for cardiac differentiation. Third, we developed an efficient approach for recording the action potentials and calcium transients simultaneously in control and patient cardiomyocytes using genetically encoded fluorescent indicators, ArcLight and R-GECO1. The dual optical recordings enabled us to observe prolonged action potentials and abnormal calcium handling in Timothy syndrome cardiomyocytes. We confirmed that roscovitine rescued the phenotypes in Timothy syndrome cardiomyocytes and that these findings were consistent with previous studies using conventional electrophysiological recordings and calcium imaging with dyes. The approaches using our optimized methods and dual optical recordings will improve iPSC applicability for disease modeling to investigate mechanisms underlying cardiac arrhythmias and to test potential therapeutics.

Laparoscopically assisted vaginal hysterectomy for women with anterior wall adherence after cesarean section.

OBJECTIVES: To share and report experiences of using lateral approach technique to perform laparoscopically assisted vaginal hysterectomy (LAVH) for women with anterior wall adherence after cesarean section. METHODS: We analyzed a retrospective chart review of 47 women with anterior wall adhesion after a cesarean section who underwent LAVH from March 1st 2003 to March 31st 2012, selected from a total of 1967 women who underwent LAVH during that period. RESULTS: The median age of the patients was 42 years (range 34-56 years). The median operating time was 120 minutes (range 85-240 minutes), and the median weight of the removed uterus was 247 g (range 50-896 g). The median change in hemoglobin level was 2.0 g/dL (range 0-3.0 g/dL). The median hospital stay was 3.0 days (range 2-6 days). There were complications in 2 cases: bladder injury in one and postoperative ileus in the other. There were no conversions to laparotomy. CONCLUSIONS: Lateral approach technique to make a pneumoperitoneum and to perform adhesiolysis is effective in LAVH for women with anterior wall adherence after cesarean section.

Laparoscopic management of pelvic splenosis.

Splenosis is defined as heterotopic autotransplantation of spleen tissue following traumatic rupture of the spleen, or surgery. It is a benign disease that is generally without any symptoms and is discovered incidentally. Surgical intervention is recommended if symptoms are present. We report the successful laparoscopic management of a 49-year-old Korean woman with splenosis-associated symptoms who had undergone splenectomy.

Surgical and obstetric outcomes of laparoscopic management for women with heterotopic pregnancy.

AIM: The aim of this study was to investigate the obstetric outcomes and clinical efficacy of laparoscopic surgery for women with heterotopic pregnancy. MATERIAL AND METHODS: We conducted a retrospective study of women who had undergone laparoscopic surgery for heterotopic pregnancy. The primary outcome was the feasibility of laparoscopic surgery for the treatment of heterotopic pregnancy and the secondary outcomes were obstetric outcomes. RESULTS: Seventeen women underwent laparoscopic surgery for heterotopic pregnancy: 14 with tubal heterotopic pregnancies and three with cornual heterotopic pregnancies. There were no intraoperative or postoperative complications. Of these women, 13 delivered 14 healthy babies, whereas two failed to maintain their pregnancies; one had a missed abortion 2 weeks after the surgery and the other had a miscarriage due to preterm premature rupture of the membrane at 16 gestational weeks. The remaining two women have ongoing pregnancies. CONCLUSION: Laparoscopic surgery performed by experienced surgeons is a feasible and beneficial surgical modality for treating heterotopic pregnancy.

Can laparoscopic radical hysterectomy be a standard surgical modality in stage IA2-IIA cervical cancer?

OBJECTIVES: To determine if laparoscopic radical hysterectomy (LRH) can be substituted for radical abdominal hysterectomy for women with International Federation of Gynecology and Obstetrics (FIGO) stage IA2-IIA cervical cancer. METHODS: We retrospectively reviewed the medical records of cervical cancer patients who underwent LRH with laparoscopic pelvic lymphadenectomy (LPL) and/or laparoscopic para-aortic lymphadenectomy (LPAL) from March 2003 to December 2011. RESULTS: Of 118 enrolled patients, six were in FIGO stage IA2, 66 were in IB1, 41 were in IB2, one was in IIA1, and four were in IIA2. The median operating time, perioperative hemoglobin change, the number of harvested pelvic and para-aortic lymph nodes were 270 min (range, 120-495), 1.7 g/dL (range, 0.1-5), 26 (range, 9-55), and 7 (range, 1-39), respectively. There was no unplanned conversion to laparotomy. Intra- and postoperative complications occurred in 16 (13.5%) and 8 (6.7%) patients, respectively. In a median follow-up of 31 months (range, 1-89), 5-year recurrence-free and overall survival rates were 90% and 89%, respectively. Univariate analysis showed that cervical stromal invasion (P=0.023) and lymph node metastasis (P=0.018) affected survival rate. Cox-proportional hazards regression analysis showed that lymph node metastasis was the only independent factor for poor prognosis (hazard ratio=7.0, P=0.022). CONCLUSIONS: LRH with LPL and/or LPAL in women with stage IA2-IIA cervical cancer is safe and feasible in terms of survival and morbidity. Our data suggest the need for larger prospective trials which could support this approach as a new standard of care for stage IA2-IIA cervical cancer.

Computerized fetal heart rate monitoring after vibroacoustic stimulation in the anencephalic fetus.

BACKGROUND: To quantify changes in fetal heart rate (FHR) parameters after vibroacoustic stimulation (VAS) and to evaluate the usefulness of VAS testing (VAST) in anencephalic fetuses. Our findings may also help to clarify the route(s) of vibration and sound transmission during VAST. STUDY DESIGN AND SUBJECTS: We obtained the antepartum FHR tracings of 16 anencephalic fetuses, including both the nonstress test (NST) and VAST. Using a computerized monitoring system, HYFM, we determined all FHR parameters from data collected for 10 min before and 10 min after VAS, at successive gestational stages. RESULTS: We observed three false reactive responses at term. The false reactive rate for VAST (3/16) was higher than that for NST (1/16). No FHR parameters increased significantly after VAS except for the number of fetal movements (FM), which increased significantly in all gestational groups (25th-32nd and 33rd-40th weeks). CONCLUSIONS: These findings call attention to an increased probability of a false reactive response in VAST analysis, when the fetus is affected by a CNS disorder. Increased numbers of FM after VAS suggest that the vibratory pathway is more likely to elicit fetal response than the auditory pathway in this setting, and that the vibratory stimulation travels by subcortical rather than by cortical pathways.

Laparoscopic ureteroureteral anastomosis for distal ureteral injuries during gynecologic laparoscopic surgery.

STUDY OBJECTIVE: To estimate the feasibility and surgical outcomes of laparoscopic ureteroureteral for treatment of distal ureteral injuries. DESIGN: Retrospective clinical study (Canadian Task Force classification II-2). SETTING: University teaching hospital. PATIENTS: Four women with ureteral transection or ureterovaginal fistula. INTERVENTION: Laparoscopic ureteroureteral . MEASUREMENTS AND MAIN RESULTS: Median age of patients was 44 (range, 33-63) years, and median operating time was 110 (range, 85-150) minutes. There were no conversions to laparotomy. No intraoperative or postoperative complications occurred. Follow-up ranged from 20 to 46 months. All patients have been asymptomatic, and follow-up intravenous pyelograms and ultrasound examinations have been normal. CONCLUSION: Laparoscopic ureteroureteral anastomosis is an alternative surgical option in women with distal ureteral injuries during gynecologic laparoscopic surgery.

Laparoscopic appendectomy performed during pregnancy by gynecological laparoscopists.

OBJECTIVE: To evaluate the safety, feasibility, and pregnancy outcomes of laparoscopic appendectomy (LA) during pregnancy. STUDY DESIGN: A retrospective review of eight pregnant women who underwent LA from January 2007 to December 2008. RESULTS: The median age of the patients and median parity were 29.5 years (range, 25-34 years) and 0 (range, 0-1), respectively. The median operating time of LA was 22.5 min (range, 15-40 min). The median length of hospital stay was 3 days (range, 2-4 days). There was no maternal or fetal mortality or morbidity, conversion to laparotomy, or uterine injury. Seven women delivered seven healthy infants. One patient chose to have an elective abortion in another hospital. The histopathological diagnoses of the resected appendices were of acute appendicitis. CONCLUSION: LA performed by gynecologic laparoscopists in pregnant women is safe, feasible, and effective.