Automated Quantitative Analysis of Shape Features in Human Epithelial Monolayers and Spheroids Generated from Colorectal Cancer Cells.

Advancements in microscopy techniques permit us to acquire endless datasets of images. A major bottleneck in cell imaging is how to analyze petabytes of data in an effective, reliable, objective, and effortless way. Quantitative imaging is becoming crucial to disentangle the complexity of many biological and pathological processes. For instance, cell shape is a summary readout of a myriad of cellular processes. Changes in cell shape use to reflect changes in growth, migration mode (including speed and persistence), differentiation stage, apoptosis, or gene expression, serving to predict health or disease. However, in certain contexts, e.g., tissues or tumors, cells are tightly packed together, and measurement of individual cellular shapes can be challenging and laborious. Bioinformatics solutions like automated computational image methods provide a blind and efficient analysis of large image datasets. Here we describe a detailed and friendly step-by-step protocol to extract various cellular shape parameters quickly and accurately from colorectal cancer cells forming either monolayers or spheroids. We envision those similar settings could be extended to other cell lines, colorectal and beyond, either label/unlabeled or in 2D/3D environments.

APC-driven actin nucleation powers collective cell dynamics in colorectal cancer cells.

Cell remodeling relies on dynamic rearrangements of cell contacts powered by the actin cytoskeleton. The tumor suppressor adenomatous polyposis coli (APC) nucleate actin filaments (F-actin) and localizes at cell junctions. Whether APC-driven actin nucleation acts in cell junction remodeling remains unknown. By combining bioimaging and genetic tools with artificial intelligence algorithms applied to colorectal cancer cell, we found that the APC-dependent actin pool contributes to sustaining levels of F-actin, as well as E-cadherin and occludin protein levels at cell junctions. Moreover, this activity preserved cell junction length and angle, as well as vertex motion and integrity. Loss of this F-actin pool led to larger cells with slow and random cell movement within a sheet. Our findings suggest that APC-driven actin nucleation promotes cell junction integrity and dynamics to facilitate collective cell remodeling and motility. This offers a new perspective to explore the relevance of APC-driven cytoskeletal function in gut morphogenesis.

E-&mHealth interventions targeting nutrition, physical activity, sedentary behavior, and/or obesity among children: A scoping review of systematic reviews and meta-analyses.

Childhood obesity is a public health concern. Electronic and mobile health (e-&mHealth) approaches can facilitate the delivery of interventions for obesity prevention and treatment. Synthesizing reviews of e-&mHealth interventions to improve weight and weight-related behaviors (physical activity, sedentary behavior, and diet) is useful to characterize the current scope of the literature and identify opportunities for future reviews and studies. Using a scoping review methodology, we aimed to evaluate the breadth and methodological quality of systematic reviews and meta-analyses of e-&mHealth interventions targeting weight and weight-related behaviors in children and adolescents aged <19 years. A systematic search of seven databases was conducted, including reviews published between 2000 and 2019. Review characteristics were extracted, and methodological quality was assessed using the AMSTAR 2 tool. Forty-five systematic reviews and meta-analyses were included. All reviews evaluated intervention efficacy (100%), but few assessed other aspects (20% in total) such as cost-effectiveness. Smartphone applications (47%), text messages (44%), and websites (35%) were the main modalities. Weight (60%), physical activity (51%), and diet (44%) were frequently assessed, unlike sedentary behavior (8%). Most reviews were rated as having critically low or low methodological quality (97%). Reviews that identify the effective active ingredients of interventions and explore metrics beyond efficacy are recommended.

Pregnant Women Have Poor Carbohydrate Knowledge and Do Not Receive Adequate Nutrition Education.

OBJECTIVES: In order to manage blood glucose levels in pregnancy, women need to know what and how much to eat, particularly for foods containing carbohydrate. The aim was to assess pregnant women's carbohydrate and standard serve size knowledge and examine whether health professionals provided nutrition education. METHODS: Between July 2017 and April 2018 Australian pregnant women were recruited to complete an online survey, including a modified PedCarbQuiz carbohydrate knowledge questionnaire and an online buffet, where they selected images equivalent to one Australian Guide to Healthy Eating (AGHE) standard serve size. RESULTS: 186 pregnant women (mean age 30.9 years, SD = 4.7 years) 12-22 weeks gestation completed the survey. Participants achieved a median score of 27/36 for identification of carbohydrate-containing foods and a median score of 1/12 (range 0-11) for identification of grams of carbohydrate in specific portions. Participants achieved a median score of 14/22 (range 4-19) for identification of one AGHE standard serve of 11 carbohydrate-containing foods. Less than half (n = 92, 49.5%) received nutrition education from health professionals. CONCLUSIONS FOR PRACTICE: Pregnant women had sub-optimal carbohydrate knowledge. This could contribute to impaired blood glucose concentrations and risk of adverse health outcomes in pregnancy. Opportunities for pregnant women to access nutrition advice from health professionals should be explored.

Embryotoxic cytokines-Potential roles in embryo loss and fetal programming.

Cytokines in the reproductive tract environment at conception mediate a dialogue between the embryo and maternal tissues to profoundly influence embryo development and implantation success. Through effects on gene expression and the cell stress response, cytokines elicit an epigenetic impact with consequences for placental development and fetal growth, which in turn affect metabolic phenotype and long-term health of offspring. There is substantial evidence demonstrating that pro-survival cytokines, such as GM-CSF, CSF1, LIF, HB-EGF and IGFII, support embryos to develop optimally. Less attention has been paid to cytokines that adversely impact embryo development, including the pro-inflammatory cytokines TNF, TRAIL and IFNG. These agents elicit cell stress, impair cell survival and retard blastocyst development, and at sufficiently high concentrations, can cause embryo demise. Experiments in mice suggest these so-called 'embryotoxic' cytokines can harm embryos through pro-apoptotic and adverse programming effects, as well as indirectly suppressing uterine receptivity through the maternal immune response. Embryotrophic factors may mitigate against and protect from these adverse effects. Thus, the balance between embryotrophic and embryotoxic cytokines can impart effects on embryo development and implantation, and has the potential to contribute to endometrial 'biosensor' function to mediate embryo selection. Embryotoxic cytokines can be elevated in plasma and reproductive tract tissues in inflammatory conditions including infection, diabetes, obesity, PCOS and endometriosis. Studies are therefore warranted to investigate whether excessive embryotoxic cytokines contribute to infertility and recurrent implantation failure in women, and compromised reproductive performance in livestock animals.

ADAMTS proteases in fertility.

The reproductive organs are unique among adult organs in that they must undergo continual tissue remodelling as a key aspect of their normal function. The processes for persistent maturation and release of new gametes, as well as fertilisation, implantation, placentation, gestation and parturition involve cyclic development and regression of tissues that must continually regenerate to support fertility. The ADAMTS family of proteases has been shown to contribute to many aspects of the tissue morphogenesis required for development and function of each of the reproductive organs. Dysregulation or functional changes in ADAMTS family proteases have been associated with reproductive disorders such as polycystic ovarian syndrome (PCOS) and premature ovarian failure (POF). Likewise, proteolytic substrates of ADAMTS enzymes have also been linked to reproductive function. New insight into the roles of ADAMTS proteases has yielded a deeper understanding of the molecular mechanisms behind fertility with clinical potential to generate therapeutic targets to resolve infertility, develop biomarkers that predict dysfunction of the reproductive organs and potentially offer targets for development of non-hormonal male and female contraceptives.

Activation of Mouse Cumulus-Oocyte Complex Maturation In Vitro Through EGF-Like Activity of Versican.

In vitro maturation of oocytes is suboptimal to in vivo maturation with altered gene expression and compromised oocyte quality. The large proteoglycan versican is abundant in mouse cumulus-oocyte complexes (COCs) matured in vivo but is absent in cultured COCs. Versican is also positively correlated with human oocyte quality. Versican contains an epidermal growth factor (EGF) motif, and based on EGF-like activities in other systems we hypothesized that versican acts as an EGF-like signaling factor during COC maturation. Here, we purified recombinant versican and compared its function with that of EGF during in vitro maturation (IVM). Versican significantly increased cumulus expansion and induced cumulus-specific genes Ptgs2, Tnfaip6, and Has2, which was blocked by EGF receptor antagonist. Microarray analysis revealed that versican has overlapping function with EGF; however, a subset of genes was uniquely altered following 6 h of IVM with either treatment. Following 6 h of IVM, both Areg and Ereg were significantly increased by both treatments, whereas Egln3, Nr4a1, Nr4a2, Nr4a3, and Adamts5 were significantly higher following versican treatment compared with EGF. In contrast, Sprr1a and Aqp3 were increased after 6 h of EGF but not versican treatment. To determine whether there were temporal differences, COCs were cultured with EGF or versican for 0-12 h. Versican-induced expression occurred later but remained elevated for longer compared with EGF for Ptgs2, Ereg, and Nr4a3. The unique expression profiles of Aqp3 and Nr4a3 during IVM were similarly regulated in vivo. These data indicate that versican has EGF-like effects on COC gene expression, but with distinct temporal characteristics.

Redox and anti-oxidant state within cattle oocytes following in vitro maturation with bone morphogenetic protein 15 and follicle stimulating hormone.

The developmental competence of cumulus oocyte complexes (COCs) can be increased during in vitro oocyte maturation with the addition of exogenous oocyte-secreted factors, such as bone morphogenetic protein 15 (BMP15), in combination with hormones. FSH and BMP15, for example, induce different metabolic profiles within COCs-namely, FSH increases glycolysis while BMP15 stimulates FAD and NAD(P)H accumulation within oocytes, without changing the redox ratio. The aim of this study was to investigate if this BMP15-induced NAD(P)H increase was due to de novo NADPH production. Cattle COCs were cultured with FSH and/or recombinant human BMP15, resulting in a significant decrease in glucose-6-phosphate dehydrogenase activity (P < 0.05). Inhibition of isocitrate dehydrogenase (IDH) during this process decreased NAD(P)H intensity threefold in BMP15-treated oocytes, suggesting that BMP15 stimulates IDH and NADPH production via the tricarboxylic acid cycle. As NADPH is a reducing agent, reduced glutathione (GSH), H2O2, and mitochondrial activity were also measured to assess the general redox status of the oocyte. FSH alone decreased GSH levels whereas the combination of BMP15 and FSH sustained higher levels. Expression of genes encoding glutathione-reducing enzymes were also lower in oocytes cultured in the presence of FSH alone. BMP15 supplementation further promoted mitochondrial localization patterns that are consistent with enhanced developmental competence. Metabolomics revealed significant consumption of glutamine and production of alanine by COCs matured with both FSH and BMP15 compared to the control (P < 0.05). Hence, BMP15 supplementation differentially modulates reductive metabolism and mitochondrial localization within the oocyte. In comparison, FSH-stimulation alone decreases the oocytes' ability to regulate cellular stress, and therefore utilizes other mechanisms to improve developmental competence.

Hemoglobin: a gas transport molecule that is hormonally regulated in the ovarian follicle in mice and humans.

An increasing number of nonerythroid tissues are found to express hemoglobin mRNA and protein. Hemoglobin is a well-described gas transport molecule, especially for O2, but also for NO, CO2, and CO, and also acts as a reactive oxygen species scavenger. We previously found Hba-a1 and Hbb mRNA and protein at high levels within mouse periovulatory cumulus cells, but not in cumulus following in vitro maturation. This led us to investigate the temporal and spatial regulation in follicular cells during the periovulatory period. Cumulus-oocyte complexes were collected from equine chorionic gonadotropin/human chorionic gonadotropin-treated peripubertal SV129 female mice and collected and analyzed for gene expression and protein localization at a variety of time points over the periovulatory period. A further cohort matured in vitro with different forms of hemoglobin (ferro- and ferrihemoglobin) under different O2 atmospheric conditions (2%, 5%, and 20% O2) were subsequently fertilized in vitro and cultured to the blastocyst stage. Murine mRNA transcripts for hemoglobin were regulated by stimulation of the ovulatory cascade, in both granulosa and cumulus cells, and expression of HBA1 and HBB was highly significant in human granulosa and cumulus, but erythrocyte cell marker genes were not. Several other genes involved in hemoglobin function were similarly luteinizing hormone-regulated, including genes for heme biosynthesis. Immunohistochemistry revealed a changing localization pattern of HBA-A1 protein in murine cumulus cells and oocytes following the ovulatory signal. Significantly, no positive staining for HBA-A1 protein was observed within in vitro-matured oocytes, but, if coincubated with ferro- or ferrihemoglobin, cytoplasmic HBA-A1 was observed, similar to in vivo-derived oocytes. Addition of ferro-, but not ferrihemoglobin, had a small, positive effect on blastocyst yield, but only under either 2% or 20% O2 gas atmosphere. The identification of hemoglobin within granulosa and cumulus cells poses many questions as to its function in these cells. There are several possible roles, the most likely of which is either an O2 or NO sequestering molecule; perhaps both roles are engaged. The strong endocrine regulation during the periovulatory period suggests to us that one potential function of hemoglobin is to provide a short-lived hypoxic environment by binding very tightly any available O2. This, in turn, facilitates the differentiation of the follicle towards corpus luteum formation by enabling the stabilization of a key transcription factor known to initiate such differentiation: hypoxia inducible factor.

Oxygen-regulated gene expression in murine cumulus cells.

Oxygen is an important component of the environment of the cumulus-oocyte complex (COC), both in vivo within the ovarian follicle and during in vitro oocyte maturation (IVM). Cumulus cells have a key role in supporting oocyte development, and cumulus cell function and gene expression are known to be altered when the environment of the COC is perturbed. Oxygen-regulated gene expression is mediated through the actions of the transcription factors, the hypoxia-inducible factors (HIFs). In the present study, the effect of oxygen on cumulus cell gene expression was examined following in vitro maturation of the murine COC at 2%, 5% or 20% oxygen. Increased expression of HIF-responsive genes, including glucose transporter-1, lactate dehydrogenase A and BCL2/adenovirus E1B interacting protein 3, was observed in cumulus cells matured at 2% or 5%, compared with 20% oxygen. Stabilisation of HIF1α protein in cumulus cells exposed to low oxygen was confirmed by western blot and HIF-mediated transcriptional activity was demonstrated using a transgenic mouse expressing green fluorescent protein under the control of a promoter containing hypoxia response elements. These results indicate that oxygen concentration influences cumulus cell gene expression and support a role for HIF1α in mediating the cumulus cell response to varying oxygen.

Comparative analyses of lung transcriptomes in patients with alveolar capillary dysplasia with misalignment of pulmonary veins and in foxf1 heterozygous knockout mice.

Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) is a developmental disorder of the lungs, primarily affecting their vasculature. FOXF1 haploinsufficiency due to heterozygous genomic deletions and point mutations have been reported in most patients with ACDMPV. The majority of mice with heterozygous loss-of-function of Foxf1 exhibit neonatal lethality with evidence of pulmonary hemorrhage in some of them. By comparing transcriptomes of human ACDMPV lungs with control lungs using expression arrays, we found that several genes and pathways involved in lung development, angiogenesis, and in pulmonary hypertension development, were deregulated. Similar transcriptional changes were found in lungs of the postnatal day 0.5 Foxf1+/- mice when compared to their wildtype littermate controls; 14 genes, COL15A1, COL18A1, COL6A2, ESM1, FSCN1, GRINA, IGFBP3, IL1B, MALL, NOS3, RASL11B, MATN2, PRKCDBP, and SIRPA, were found common to both ACDMPV and Foxf1 heterozygous lungs. Our results advance knowledge toward understanding of the molecular mechanism of ACDMPV, lung development, and its vasculature pathology. These data may also be useful for understanding etiologies of other lung disorders, e.g. pulmonary hypertension, bronchopulmonary dysplasia, or cancer.

Macrophages regulate corpus luteum development during embryo implantation in mice.

Macrophages are prominent in the uterus and ovary at conception. Here we utilize the Cd11b-Dtr mouse model of acute macrophage depletion to define the essential role of macrophages in early pregnancy. Macrophage depletion after conception caused embryo implantation arrest associated with diminished plasma progesterone and poor uterine receptivity. Implantation failure was alleviated by administration of bone marrow-derived CD11b+F4/80+ monocytes/macrophages. In the ovaries of macrophage-depleted mice, corpora lutea were profoundly abnormal, with elevated Ptgs2, Hif1a, and other inflammation and apoptosis genes and with diminished expression of steroidogenesis genes Star, Cyp11a1, and Hsd3b1. Infertility was rescued by exogenous progesterone, which confirmed that uterine refractoriness was fully attributable to the underlying luteal defect. In normally developing corpora lutea, macrophages were intimately juxtaposed with endothelial cells and expressed the proangiogenic marker TIE2. After macrophage depletion, substantial disruption of the luteal microvascular network occurred and was associated with altered ovarian expression of genes that encode vascular endothelial growth factors. These data indicate a critical role for macrophages in supporting the extensive vascular network required for corpus luteum integrity and production of progesterone essential for establishing pregnancy. Our findings raise the prospect that disruption of macrophage-endothelial cell interactions underpinning corpus luteum development contributes to infertility in women in whom luteal insufficiency is implicated.

Development and hormonal regulation of the ovarian lymphatic vasculature.

The lymphatic vasculature plays a number of essential physiological roles including maintaining fluid homeostasis, providing a network for the transport of immune cells, and facilitating the uptake of fat-soluble nutrients from the gastrointestinal tract. Although the critical importance and remodeling capacity of the blood vasculature has been well described within the ovary, just a few reports describe the lymphatic vasculature. Using histological and molecular techniques, we report the kinetics of ovarian lymphangiogenesis and the hormonal regulation of lymphangiogenic growth factors associated with key stages of ovarian follicle growth. We exploited the Adamts1-null mouse model, a model with a previously characterized lymphatic defect to further interrogate the mechanisms controlling ovarian lymphangiogenesis. The establishment and development of the ovarian lymphatic vascular network in postnatal developing ovaries was associated with the presence and hormonal regulation of the lymphangiogenic growth factors and their receptors, including Vegfc, Vegfd, and Vegfr3. We characterized the hormonally regulated remodeling of the ovarian lymphatic vasculature in response to FSH and estradiol. The lymphatic network was defective in the Adamts1-null ovary, clearly demonstrating both the involvement of FSH/estradiol and the Adamts1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) protease in ovarian lymphangiogenesis. This study provides the first evidence of a malleable lymphatic system responsive to hormonal changes of the female reproductive cycle, at least in the mouse ovary, suggesting a role for lymphatic vessel functions in normal folliculogenesis.