FOXA1/2 depletion drives global reprogramming of differentiation state and metabolism in a human liver cell line and inhibits differentiation of human stem cell-derived hepatic progenitor cells.

FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA factors dictate human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in controlling hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA mediated knockdown of FOXA1/2 in HepG2 cells significantly downregulated albumin (p < .05) and GRN TF gene expression (HNF4α, HEX, HNF1ß, TBX3) (p < .05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid [GSC], FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < .05), all consistent with partial/transient reprograming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility of phenotype. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, with concomitant accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During human pluripotent stem cells (hPSC)-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate that FOXA1/2 controls hepatic and developmental GRN, and their knockdown leads to reprogramming of both differentiation and metabolism, with applications in studies of cancer, differentiation, and organogenesis.

Determination of Whole Blood Loss From Minimally Invasive Myomectomy Using a Standardized Formula: A Pilot Study.

STUDY OBJECTIVE: To determine the median perioperative blood loss (PBL) during minimally invasive surgical (MIS) myomectomy. DESIGN: Prospective pilot study. SETTING: Large academic teaching hospital. PATIENTS: Thirty-one patients underwent laparoscopic or robotic myomectomy and completed a postoperative complete blood count (CBC) from November 2020 to August 2022. Patients had to have at least one fibroid greater than or equal to 3 cm on preoperative imaging. INTERVENTIONS: A CBC was collected preoperatively within 7 days of surgery. Estimated blood loss (EBL) was determined by the surgeon intraoperatively. A repeat CBC was drawn between postoperative days 2 through 4. PBL was calculated using the equation PBL = (patient weight in kg × 65 cc/kg) × (preoperative hematocrit - postoperative hematocrit)/preoperative hematocrit. MEASUREMENTS AND MAIN RESULTS: Median PBL (536.3 cc [270.0, 909.3]) was greater than median EBL (200.0 cc [75.0, 500.0]). PBL ranged from a net gain of 191.5 cc to net loss of 2362.5 cc. Median size of the largest fibroid on preoperative imaging was 8.8 cm (6.6, 11.5), and median weight of fibroids removed was 321 g (115, 519). About half of patients (51.6%) had one fibroid removed, and 48.4% had 2 or more fibroids removed. Five patients were converted to laparotomy, 4 from robotic approaches. Two patients required a blood transfusion. CONCLUSION: Calculated PBL was greater than intraoperative EBL. This suggests there is continued blood loss post myometrial bed closure. Blood loss should be evaluated both during and after myomectomy, as intraoperative EBL underestimates total PBL.

A Randomized Multicenter Study Assessing the Educational Impact of a Computerized Interactive Hysterectomy Trainer on Gynecology Residents.

STUDY OBJECTIVE: To assess the improvement of cognitive surgical knowledge of laparoscopic hysterectomy in postgraduate year (PGY) 1 and 2 gynecology residents who used an interactive computer-based Laparoscopic Hysterectomy Trainer (Red Llama, Inc., Seattle, WA). DESIGN: A multicenter, randomized, controlled study (Canadian Task Force classification I). SETTING: Five departments of obstetrics and gynecology: Keck School of Medicine of the University of Southern California, Los Angeles, CA; University of California, Los Angeles, Los Angeles, CA; University of Washington, Seattle, WA; University of British Columbia, Vancouver, British Columbia, Canada; and University of Toronto, Toronto, Ontario, Canada. PARTICIPANTS: Gynecology residents, fellows, faculty, and minimally invasive surgeons. INTERVENTIONS: The use of an interactive computer-based Laparoscopic Hysterectomy Trainer. MEASUREMENTS AND MAIN RESULTS: In phase 1 of this 3-phase multicenter study, 2 hysterectomy knowledge assessment tests (A and B) were developed using a modified Delphi technique. Phase 2 administered these 2 online tests to PGY 3 and 4 gynecology residents, gynecology surgical fellows, faculty, and minimally invasive surgeons (n = 60). In phase 3, PGY 1 and 2 gynecology residents (n = 128) were recruited, and 101 chose to participate, were pretested (test A), and then randomized to the control or intervention group. Both groups continued site-specific training while the intervention group additionally used the Laparoscopic Hysterectomy Trainer. Participant residents were subsequently posttested (test B). Phase 2 results showed no differences between cognitive tests A and B when assessed for equivalence, internal consistency, and reliability. Construct validity was shown for both tests (p < .001). In phase 3, the pretest mean score for the control group was 242 (standard deviation [SD] = 56.5), and for the intervention group it was 217 (SD = 57.6) (nonsignificant difference, p = .089). The t test comparing the posttest control group (mean = 297, SD = 53.6) and the posttest intervention group (mean = 343, SD = 50.9) yielded a significant difference (p < .001, 95% confidence interval, 48.4-108.8). Posttest scores for the intervention group were significantly better than for the control group (p < .001). CONCLUSION: Using the Laparoscopic Hysterectomy Trainer significantly increased knowledge of the hysterectomy procedure in PGY 1 and 2 gynecology residents.

Video-based peer feedback through social networking for robotic surgery simulation: a multicenter randomized controlled trial.

OBJECTIVE: To examine the feasibility and outcomes of video-based peer feedback through social networking to facilitate robotic surgical skill acquisition. BACKGROUND: The acquisition of surgical skills may be challenging for novel techniques and/or those with prolonged learning curves. METHODS: Randomized controlled trial involving 41 resident physicians performing the Tubes (Da Vinci Intuitive Surgical, Sunnyvale, CA) simulator exercise with versus without peer feedback of video-recorded performance through a social networking Web page. Data collected included simulator exercise score, time to completion, and comfort and satisfaction with robotic surgery simulation. RESULTS: There were no baseline differences between the intervention group (n = 20) and controls (n = 21). The intervention group showed improvement in mean scores from session 1 to sessions 2 and 3 (60.7 vs 75.5, P < 0.001, and 60.7 vs 80.1, P < 0.001, respectively). The intervention group scored significantly higher than controls at sessions 2 and 3 (75.5 vs 59.6, P = 0.009, and 80.1 vs 65.9, P = 0.019, respectively). The mean time (seconds) to complete the task was shorter for the intervention group than for controls during sessions 2 and 3 (217.4 vs 279.0, P = 0.004, and 201.4 vs 261.9, P = 0.006, respectively). At the study conclusion, feedback subjects were more comfortable with robotic surgery than controls (90% vs 62%, P = 0.021) and expressed greater satisfaction with the learning experience (100% vs 67%, P = 0.014). Of the intervention subjects, 85% found that peer feedback was useful and 100% found it effective. CONCLUSIONS: Video-based peer feedback through social networking appears to be an effective paradigm for surgical education and accelerates the robotic surgery learning curve during simulation.

Intravascular tissue reactions induced by various types of bioabsorbable polymeric materials: correlation between the degradation profiles and corresponding tissue reactions.

INTRODUCTION: Several different bioabsorbable polymeric coil materials are currently used with the goal of improving treatment outcomes of endovascular embolization of intracranial aneurysms. However, little is known about the correlation between polymer degradation profiles and concomitant tissue responses in a blood vessel. The authors describe in vitro degradation characteristics of nine different polymeric materials and their corresponding tissue responses induced in rabbit carotid arteries. METHODS: Mass loss and molecular weight loss of nine commercially available bioabsorbable sutures were evaluated in vitro up to16 weeks. The same nine materials, as well as platinum coils, were implanted into blind-end carotid arteries (n = 44) in rabbits, and their tissue reactions were evaluated histologically 14 days after the implantation. RESULTS: Five of the nine polymers elicited moderate to strong tissue reactions relative to the remaining materials. While polymer mass loss did not correlate with their histologic findings, polymers that showed a faster rate of molecular weight loss had a tendency to present more active tissue reactions such as strong fibrocellular response around the implanted material with a moderate inflammatory cell infiltration. Maxon exhibited the fastest rate of molecular weight loss and poly-l-lactic acid the slowest. CONCLUSIONS: The rate of molecular weight loss may be an important factor that is associated with the degree of bioactivity when bioabsorbable polymers are implanted into blood vessels. For further quantitative analysis, additional experiments utilizing established aneurysm models need to be conducted.

Thrombus organization and healing in the swine experimental aneurysm model. Part I. A histological and molecular analysis.

OBJECT: The authors describe the process of thrombus organization in the swine surgical aneurysm model. METHODS: Lateral carotid artery aneurysms with immediately induced thrombosis were created in 31 swine for a time-course study. Aneurysms were evaluated at 1, 3, 7, 14, 30, and 90 days after they were created. Histological analyses included quantitative immunohistochemical studies and evaluation of collagen deposition. Complementary DNA microarray analysis was performed for gene expression profiling. The lists of up- and downregulated genes were cross-matched with lists of genes known to be associated with cytokines or the extracellular matrix. The expression of selected genes was quantified using real-time polymerase chain reaction. Functional clustering was performed with the Expression Analysis Systematic Explorer (EASE) bioinformatics package. RESULTS: Histological analysis demonstrated leukocyte and macrophage infiltration in the thrombus at Day 3, myofibroblast infiltration at Days 7 to 14, and progressive collagen deposition and contraction thereafter. Tissue organization occurred in a centripetal fashion. A previously undescribed reticular network of connective tissue was observed at the periphery of the aneurysm at Day 3. Macrophages appeared critical to this thrombus organization. A total of 1109 genes were significantly changed from reference time zero during the time course: CXCL14, which produces a monocyte-specific chemokine, was upregulated over 100-fold throughout the time course; IGF1 was upregulated fourfold at Day 7, whereas IGFBP2 was downregulated approximately 50% at Days 7 and 14. Osteopontin (SPP1) upregulation increased from 30-fold at Day 30 to 45-fold at Day 14. The EASE analysis yielded eight functional classes of gene expression. CONCLUSIONS: This investigation provides a detailed histological and molecular analysis of thrombus organization in the swine aneurysm model. The companion study will describe the effect of embolic bioabsorbable polymers on this process.

Thrombus organization and healing in an experimental aneurysm model. Part II. The effect of various types of bioactive bioabsorbable polymeric coils.

OBJECT: Bioabsorbable polymeric material coils are being used in the endovascular treatment of aneurysms to achieve better thrombus organization than is possible using bare platinum coils. We used immunohistochemical and molecular biological analysis techniques in experimental aneurysms implanted with three different bioabsorbable polymer coils and platinum coils. METHODS: The degradation kinetics of nine polymer candidates for further analysis were first analyzed in vitro, and three materials with different degradation rates were selected. Seventy-four aneurysms were created in 37 swine using the venous pouch technique. The aneurysms were surgically implanted with one of the materials as follows (time points = 3, 7, and 14 days): Group 1, Guglielmi detachable coils (platinum); Group 2, Polysorb (90:10 polyglycolic acid [PGA]/polylactic acid); Group 3, Maxon (PGA/trimethylene carbonate); and Group 4, poly-l-lactic acid. Histological, immunohistochemical, and cDNA microarray analyses were performed on tissue specimens. RESULTS: Groups 1 and 4 showed minimal inflammatory response adjacent to the coil mass. In Group 2, Polysorb elicited a unique, firm granulation tissue that accelerated intraaneurysmal thrombus organization. In Group 3 intermediate inflammatory reactions were seen. Microarray analysis with Expression Analysis Sytematic Explorer software showed functional-cluster-gene activation to be increased at Day 7, preceding the histologic manifestation of polymer-induced granulation tissue at Day 14. A profile of expression changes in cytokine-related and extracellular membrane-related genes was compiled. CONCLUSIONS: Degradation speed was not the only factor determining the strength of the biological response. Polysorb induced an early, unique granulation tissue that conferred greater mechanical strength to the intraaneurysmal coilthrombus complex. Enhancing the formation of this polymer-induced granulation tissue may provide a new direction for improving long-term anatomical outcomes in cases involving aneurysms embolized with detachable coils.

Insulin signaling pathways in cortical dysplasia and TSC-tubers: tissue microarray analysis.

To evaluate the possible roles of the Akt/PKB-mTOR-p70S6K-S6 and cap-dependent translation (eIF4G) pathways in the pathogenesis of tuberous sclerosis complex (TSC)-associated cortical tubers and focal cortical dysplasia (FCD), we performed qualitative and semiquantitative immunohistochemical evaluation on surgically resected corticectomy specimens to detect phosphorylated molecules as activated downstream targets of the signaling pathways. A tissue microarray paraffin block was constructed from 63 archival specimens of surgically resected TSC tubers, FCDs with balloon cells, cortical dysplasia without balloon cells, and histologically normal-appearing neocortex obtained from cases with Rasmussen encephalitis, cystic-gliotic encephalopathy, and temporal lobe epilepsy. Abnormal neuroglial cells were positive for phospho-S6 and phospho-eIF4G with various staining intensities in FCDs and TSC tubers. Both proteins were much less abundantly expressed in normal-appearing neocortex. Phospho-mTOR expression was observed in neurons in all groups. The expression of phospho-S6 and phospho-eIF4G was associated with dysplastic lesions (p < 0.05), and the cytoplasmic phospho-p70S6K expression was most specific for and abundant in TSC tubers and much less prominent in other groups (p < 0.01). These results suggest that constitutive activation of cytoplasmic p70S6K plays a pivotal role in the pathogenesis of TSC tubers and that FCDs possess a distinct mechanism for activation of S6 and eIF4G.