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has been reviewed by the Editorial Board and by special expert referees. Although it is judged not acceptable for publication in Obstetrics & Gynecology in its present form, we would be willing to give further consideration to a revised version. If you wish to consider revising your manuscript, you will first need to study carefully the enclosed reports submitted by the referees and editors. Each point raised requires a response, by either revising your manuscript or making a clear and convincing argument as to why no revision is needed. To facilitate our review, we prefer that the cover letter include the comments made by the reviewers and the editor followed by your response. The revised manuscript should indicate the position of all changes made. We suggest that you use the "track changes" feature in your word processing software to do so (rather than strikethrough or underline formatting).

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Histological examination of biopsies from the normal-appearing cecum and terminal ileum is useful in a small but significant number of patients with colonic tuberculosis.

Drug resistance remains a major clinical problem despite advances in targeted therapies. In recent years, methods to culture cancer cells in three-dimensional (3D) environments to better mimic native tumors have gained increasing popularity. Nevertheless, unlike traditional two-dimensional (2D) cell cultures, analysis of 3D cultures is not straightforward. Most biochemical assays developed for 2D cultures have to be optimized for use with 3D cultures. We addressed this important problem by presenting a simple method of quantitative size-based analysis of growth and drug responses of 3D cultures of cancer cells as tumor spheroids. We used an aqueous two-phase system to form consistently sized tumor spheroids of colorectal cancer cells. Using spheroid images, we computed the size of spheroids over time and demonstrated that growth of spheroids from this analysis strongly correlates with that using a PrestoBlue biochemical assay optimized for 3D cultures. Next, we cyclically treated the tumor spheroids with a MEK inhibitor, trametinib, for 6-day periods with a recovery phase in between. This inhibitor was selected because of mutation of colon cancer cells in the MEK/ERK pathway. We used size measurements to evaluate the efficacy of trametinib and predict development of resistance of colon cancer cells during the cyclical treatment and recovery regimen. This size-based analysis closely matched the biochemical analysis of drug responses of spheroids. We performed molecular analysis and showed that resistance to trametinib emerged due to feedback activation of the PI3K/AKT signaling pathway. Therefore, we combined trametinib with a PI3K/AKT inhibitor, dactolisib, and demonstrated that size-based analysis of spheroids reliably allowed quantifying the effect of the combination treatment to prevent drug resistance. This study established that size measurements of spheroids can be used as a straightforward method for quantitative studies of drug responses of tumor spheroids and identifying drug combinations that block resistance.

BackgroundCell migration and invasion are essential processes for metastatic dissemination of cancer cells. Significant progress has been made in developing new therapies against oncogenic signaling to eliminate cancer cells and shrink tumors. However, inherent heterogeneity and treatment-induced adaptation to drugs commonly enable subsets of cancer cells to survive therapy. In addition to local recurrence, these cells escape a primary tumor and migrate through the stroma to access the circulation and metastasize to different organs, leading to an incurable disease. As such, therapeutics that block migration and invasion of cancer cells may inhibit or reduce metastasis and significantly improve cancer therapy. This is particularly more important for cancers, such as triple negative breast cancer, that currently lack targeted drugs.MethodsWe used cell migration, 3D invasion, zebrafish metastasis model, and phosphorylation analysis of 43 protein kinases in nine triple negative breast cancer (TNBC) cell lines to study effects of fisetin and quercetin on inhibition of TNBC cell migration, invasion, and metastasis.ResultsFisetin and quercetin were highly effective against migration of all nine TNBC cell lines with up to 76 and 74% inhibitory effects, respectively. In addition, treatments significantly reduced 3D invasion of highly motile TNBC cells from spheroids into a collagen matrix and their metastasis in vivo. Fisetin and quercetin commonly targeted different components and substrates of the oncogenic PI3K/AKT pathway and significantly reduced their activities. Additionally, both compounds disrupted activities of several protein kinases in MAPK and STAT pathways. We used molecular inhibitors specific to these signaling proteins to establish the migration-inhibitory role of the two phytochemicals against TNBC cells.ConclusionsWe established that fisetin and quercetin potently inhibit migration of metastatic TNBC cells by interfering with activities of oncogenic protein kinases in multiple pathways.

Hemolysis predisposes to adverse pregnancy outcomes. Yet, there are limited data on hemolysis in hypertensive disorders of pregnancy other than hemolysis, elevated liver enzymes, and low platelet count syndrome. To evaluate the prevalence and impact of hemolysis in hypertensive disorders of pregnancy, we performed a retrospective cohort study at a single center (October 2013-May 2017), among women screened for hemolysis using lactate dehydrogenase (LDH) levels. We compared LDH levels by hypertensive disorder (chronic hypertension, gestational hypertension, preeclampsia, and preeclampsia with severe features) and evaluated impact on adverse pregnancy outcomes. Data were analyzed by χ or test, ANOVA, test of medians, and logistic regression. Among 8645 deliveries, 1188 (13.7%) had a hypertensive disorder. Of these, 812 (68.4%) had LDH measurement before delivery: chronic hypertension (n=152); gestational hypertension (n=209); preeclampsia (n=216); and preeclampsia with severe features (n=235). LDH ≥400 U/L (≥1.6× normal) was more common in preeclampsia with severe features compared with other hypertensive disorders of pregnancy (9.8% versus 2.3%;<0.001); adjusted odds ratio 4.52 (95% confidence interval, 2.2-9.2; <0.001). LDH ≥400 U/L was associated with adverse maternal outcomes (41.7% versus 15.3%; <0.001), adjusted odds ratio 3.05 (95% confidence interval, 1.4-6.7; =0.006), and adverse neonatal outcomes (eg, preterm birth 59.4% versus 22.5%;<0.001). We find that elevated LDH levels are associated with adverse maternal and neonatal outcomes in hypertension and preeclampsia, independent of hemolysis, elevated liver enzymes, and low platelet count syndrome. Therefore, elevated LDH levels (≥1.6× normal or ≥400 U/L) may be considered a severe feature of preeclampsia.