The Gut Microbiome and Symptom Burden After Kidney Transplantation: An Overview and Research Opportunities.

Many kidney transplant recipients continue to experience high symptom burden despite restoration of kidney function. High symptom burden is a significant driver of quality of life. In the post-transplant setting, high symptom burden has been linked to negative outcomes including medication non-adherence, allograft rejection, graft loss, and even mortality. Symbiotic bacteria (microbiota) in the human gastrointestinal tract critically interact with the immune, endocrine, and neurological systems to maintain homeostasis of the host. The gut microbiome has been proposed as an underlying mechanism mediating symptoms in several chronic medical conditions including irritable bowel syndrome, chronic fatigue syndrome, fibromyalgia, and psychoneurological disorders via the gut-brain-microbiota axis, a bidirectional signaling pathway between the enteric and central nervous system. Post-transplant exposure to antibiotics, antivirals, and immunosuppressant medications results in significant alterations in gut microbiota community composition and function, which in turn alter these commensal microorganisms' protective effects. This overview will discuss the current state of the science on the effects of the gut microbiome on symptom burden in kidney transplantation and future directions to guide this field of study.

Mediterranean Diet Adherence and Depressive Symptoms among a Nationally Representative Sample of Pregnant Women in the United States.

BACKGROUND: Prenatal depression affects ∼12% of pregnant women in the United States and is associated with an increased risk of adverse birth outcomes and maternal mortality. Adherence to a healthy dietary pattern may reduce and/or protect against depressive symptoms. OBJECTIVES: To investigate the relationship between adherence to a Mediterranean diet and depressive symptoms among pregnant women in the United States. METHODS: We used data from the National Health and Nutrition Examination Survey (2005-2018, N = 540) and included pregnant women aged 18-44 y with a positive urine pregnancy test. The Mediterranean diet score (aMED) was calculated from 1 24-h recall; aMED typically ranges from 0-9, but in these analyses, it ranged from 0-8 because alcohol was not included. The aMED score was dichotomized as high (>3) compared with low (≤3). The Patient Health Questionnaire-9 (PHQ-9), which measures depressive symptoms, was dichotomized as lower compared with higher (PHQ-9 score ≥10), based on the clinical cutoff for patient referral. Our primary model employed logistic regression to investigate the association between aMED adherence and high depressive symptoms when controlling for socio-demographics (age, racial/ethnicity, education, poverty, and relationship status), total calories, and prepregnancy body mass index (kg/m2). We also modeled the PHQ-9 score as a continuous variable using a random-effects model. RESULTS: About 5% of pregnant women had moderate to severe depressive symptoms, and 45% were highly adherent to a Mediterranean diet. Higher adherence to a Mediterranean diet was associated with lower odds of depressive symptoms (odds ratio: 0.31, 95% confidence interval: 0.10, 0.98). Results were not significant for the continuous PHQ-9 score (ß: -0.30; 95% confidence interval: -0.90, 0.30). CONCLUSIONS: Adherence to a Mediterranean diet may have the potential to lower depressive symptoms among pregnant women; however, these results should be interpreted with caution. Nevertheless, considering the public health significance of promoting mental wellness among pregnant women, this relationship merits further examination using experimental designs.

Exploring the Effects of a Mediterranean Diet and Weight Loss on the Gut Microbiome and Cognitive Performance in Older, African American Obese Adults: A Post Hoc Analysis.

African American adults have a higher prevalence of Alzheimer's dementia (AD) than non-Hispanic Whites. The impact of a Mediterranean Diet (Med Diet) and intentional weight loss (IWL) on the gut microbiome may alter AD risk. A post hoc analysis of the Building Research in Diet and Cognition (BRIDGE) trial was performed to determine whether participation in an 8-month Med Diet lifestyle intervention with (n = 35) or without IWL (n = 31) was associated with changes in gut microbiota structure, abundance, and function and whether these changes were related to changes in cognitive performance. The results showed that family and genus alpha diversity increased significantly in both groups combined (p = 0.0075 and p = 0.024, respectively). However, there were no other significant microbially related within- or between-group changes over time. Also, an increase in Med Diet adherence was significantly associated with a decrease in alpha diversity at the phylum level only (p = 0.049). Increasing alpha diversity was associated with decreasing cognitive performance, but this association was attenuated after controlling for Med Diet adherence. In sum, an 8-month Med Diet lifestyle intervention with or without IWL did not appreciably alter the gut microbiome.

Design of the Building Research in CRC prevention (BRIDGE-CRC) trial: a 6-month, parallel group Mediterranean diet and weight loss randomized controlled lifestyle intervention targeting the bile acid-gut microbiome axis to reduce colorectal cancer risk among African American/Black adults with obesity.

BACKGROUND: Among all racial/ethnic groups, people who identify as African American/Blacks have the second highest colorectal cancer (CRC) incidence in the USA. This disparity may exist because African American/Blacks, compared to other racial/ethnic groups, have a higher prevalence of risk factors for CRC, including obesity, low fiber consumption, and higher intakes of fat and animal protein. One unexplored, underlying mechanism of this relationship is the bile acid-gut microbiome axis. High saturated fat, low fiber diets, and obesity lead to increases in tumor promoting secondary bile acids. Diets high in fiber, such as a Mediterranean diet, and intentional weight loss may reduce CRC risk by modulating the bile acid-gut microbiome axis. The purpose of this study is to test the impact of a Mediterranean diet alone, weight loss alone, or both, compared to typical diet controls on the bile acid-gut microbiome axis and CRC risk factors among African American/Blacks with obesity. Because weight loss or a Mediterranean diet alone can reduce CRC risk, we hypothesize that weight loss plus a Mediterranean diet will reduce CRC risk the most. METHODS: This randomized controlled lifestyle intervention will randomize 192 African American/Blacks with obesity, aged 45-75 years to one of four arms: Mediterranean diet, weight loss, weight loss plus Mediterranean diet, or typical diet controls, for 6 months (48 per arm). Data will be collected at baseline, mid-study, and study end. Primary outcomes include total circulating and fecal bile acids, taurine-conjugated bile acids, and deoxycholic acid. Secondary outcomes include body weight, body composition, dietary change, physical activity, metabolic risk, circulating cytokines, gut microbial community structure and composition, fecal short-chain fatty acids, and expression levels of genes from exfoliated intestinal cells linked to carcinogenesis. DISCUSSION: This study will be the first randomized controlled trial to examine the effects of a Mediterranean diet, weight loss, or both on bile acid metabolism, the gut microbiome, and intestinal epithelial genes associated with carcinogenesis. This approach to CRC risk reduction may be especially important among African American/Blacks given their higher risk factor profile and increased CRC incidence. TRIAL REGISTRATION: ClinicalTrials.gov NCT04753359 . Registered on 15 February 2021.

Gestational Insulin Resistance Is Mediated by the Gut Microbiome-Indoleamine 2,3-Dioxygenase Axis.

BACKGROUND & AIMS: Normal gestation involves a reprogramming of the maternal gut microbiome (GM) that contributes to maternal metabolic changes by unclear mechanisms. This study aimed to understand the mechanistic underpinnings of the GM-maternal metabolism interaction. METHODS: The GM and plasma metabolome of CD1, NIH-Swiss, and C57 mice were analyzed with the use of 16S rRNA sequencing and untargeted liquid chromatography-mass spectrometry throughout gestation. Pharmacologic and genetic knockout mouse models were used to identify the role of indoleamine 2,3-dioxygenase (IDO1) in pregnancy-associated insulin resistance (IR). Involvement of gestational GM was studied with the use of fecal microbial transplants (FMTs). RESULTS: Significant variation in GM alpha diversity occurred throughout pregnancy. Enrichment in gut bacterial taxa was mouse strain and pregnancy time point specific, with the species enriched at gestation day 15/19 (G15/19), a point of heightened IR, being distinct from those enriched before or after pregnancy. Metabolomics revealed elevated plasma kynurenine at G15/19 in all 3 mouse strains. IDO1, the rate-limiting enzyme for kynurenine production, had increased intestinal expression at G15, which was associated with mild systemic and gut inflammation. Pharmacologic and genetic inhibition of IDO1 inhibited kynurenine levels and reversed pregnancy-associated IR. FMT revealed that IDO1 induction and local kynurenine level effects on IR derive from the GM in both mouse and human pregnancy. CONCLUSIONS: GM changes accompanying pregnancy shift IDO1-dependent tryptophan metabolism toward kynurenine production, intestinal inflammation, and gestational IR, a phenotype reversed by genetic deletion or inhibition of IDO1. (Gestational Gut Microbiome-IDO1 Axis Mediates Pregnancy Insulin Resistance; EMBL-ENA ID: PRJEB45047. MetaboLights ID: MTBLS3598).

Using self-report RDoC measures to identify transdiagnostic translational targets for perinatal affective disorders.

Perinatal depression affects 6.5-12.9% of women, with high rates in women of color and comorbid perinatal anxiety in up to 50% of cases. The Research Domain Criteria (RDoC) provides a translational framework for identifying transdiagnostic psychiatric symptoms, but its application in perinatal affective disorders (PNAD) is yet limited. Here, we identified RDoC-based transdiagnostic features of PNAD in 140 primarily low-income Black and Hispanic women at 272 total longitudinal visits across the perinatal period. Women completed RDoC self-report measures of potential threat and reward valuation-Behavioral Inhibition System/Behavioral Activation System scale (BIS/BAS) and Intolerance of Uncertainty Scale (IUS)-and measures of depression (Patient Health Questionnaire-9; PHQ-9) and anxiety (Generalized Anxiety Disorder-7; GAD-7). Longitudinal mixed effects models assessed associations of between-person ("trait-like") and within-person ("state-like") measures of potential threat (BIS/IUS) and reward valuation (BAS-Drive) with depression and anxiety symptoms. Higher "trait-like" BIS (standardized b = 2.33, p < .001) and IUS (b = 2.97, p < .001) scores, higher "state-like" BIS (b = .71, p < .001), and lower "state-like" BAS-Drive (b = - .58, p = .04) scores were associated with worse depressive symptoms. Higher "trait-like" BIS (b = 2.22, p < .001) and IUS (b = 2.73, p < .001) and higher "state-like" BIS scores (b = .92, p < .001) were associated with worse anxiety symptoms. Potential threat may be a prominent, transdiagnostic feature of perinatal anxiety and depression, whereas reward valuation may be a non-transdiagnostic, weaker feature of perinatal depression. Potential threat is important as both a "trait-like" feature that is sustained across the perinatal period and a "state-like" feature that varies within a woman across pregnancy. Grounded in RDoC, this work reveals neurobiological targets for translational research into PNAD.

Neuroactive steroids and depression in early pregnancy.

Progesterone (P4) can be metabolized to two general classes of neuroactive steroids (NAS) -those like allopregnanolone (ALLO) and pregnanolone (PA) which are positive allosteric modulators of the Gamma Aminobutyric Acid type A (GABAA) receptor and those like isoallopregnanolone (ISOALLO) and epipregnanolone (EPI) which are negative allosteric modulators of the GABAA receptor. While exogenous administration of ALLO is effective in treating postpartum depression, knowledge gaps remain in the dynamic interplay of NAS across the perinatal period. In particular little is known about ALLO and PA in relation to depression earlier in pregnancy, and the role of ISOALLO and EPI in relation to depression at any point in the perinatal period. In a prospective, nested case/control study in low-income women of color, we compared the metabolism of P4 to four NAS (i.e., ratios ALLO:P4, PA:P4, ISOALLO:P4, EPI:P4) in pregnant women with depression at either or both of the first and second trimesters (cases) and women without depression at either time point (controls). Fifty women (36% depressed, 56% Black, 28% Latina) completed depression screening using a computerized adaptive test of mental health (CAT-MH™) and provided blood serum samples in both trimesters. In longitudinal mixed effects models of both trimesters, PND cases showed higher ratios of ALLO:P4 (p = .002) and PA:P4 (p = .03) compared to controls. In regression models of only first trimester data, there was no significant difference in NAS ratios between cases and controls (p > .05). Conversely, in models of the second trimester, ratios of PA:P4 (p = .002) and ISOALLO:P4 (p = .01) were significantly higher in cases compared to controls, and ratios of ALLO:P4 (p = .08) and EPI:P4 (p = .1) also trended higher in cases. The most severe cases, those with depression at both trimesters, showed an increase in ALLO:P4 (p = .06) and EPI:P4 (p < .001) ratios from the first to the second trimester, whereas controls showed a decrease in these ratios. Secondary analyses confirmed higher levels of ALLO (p = .04) and PA (p = .07) overall in cases compared to controls, along with higher levels of PA (p = .005) and ISOALLO (p = .02) in the second trimester alone. This work suggests a dynamic relationship between NAS and PND; whereas low ALLO levels have been previously associated with postpartum depression, earlier in pregnancy a higher metabolism of P4 to ALLO (and higher ALLO levels) is associated with depression. Some women may show a hormone-sensitive depressive response to acute increases in NAS metabolism in early pregnancy.

Capitalizing on transcriptome profiling to optimize and identify targets for promoting early murine folliculogenesis in vitro.

In vitro ovarian follicle culture is an active area of research towards providing fertility options for survivors of childhood cancer. Late-stage murine follicles (multilayer secondary and onwards) can be cultured successfully to maturity to obtain a meiotically competent oocyte for fertilization, but primordial and primary follicles usually die in culture because many key components of early follicle development are still unknown and difficult to mimic in vitro. To engineer a biomimetic three-dimensional culture system with high efficacy and reproducibility for the clinic, detailed mechanisms of early folliculogenesis must be uncovered. Previous studies have shown that primary murine follicles co-cultured in groups, in contrast to single follicles cultured in isolation, can reach preovulatory size and produce competent oocytes, but the factors accounting for the synergy of follicle co-culture are still unknown. To probe the underlying mechanisms of successful follicle co-culture, we conducted a time-course experiment for murine follicles encapsulated in 0.3% alginate hydrogels and compared between two conditions: groups of 5 (5X) versus groups of 10 (10X). For every 2 days during the course of 12 days, follicles were dissociated and somatic cells were isolated for microarray-based gene expression analysis (n = 380 follicles for 5X and n = 430 follicles for 10X). Gene activities in follicles co-cultured in larger groups (10X) had a distinct transcriptomic profile of key genes and pathways such as prolactin signaling and angiogenesis-related genes when compared to cells from follicles co-cultured in the smaller cohort (5X). To benchmark the results for follicles grown in culture, we compared our microarray data to data from murine follicles freshly isolated from the ovary at comparable stages of development previously published by Bernabé et al. Comparison of these datasets identified similarities and differences between folliculogenesis in the native microenvironment and the engineered in vitro system. A more detailed understanding of follicle growth in vitro will not only allow for better culture methods but also advance the field towards providing improved fertility options for survivors of childhood cancer.

The Role of the Gut Microbiota in the Prevention and Management of Gestational Diabetes Mellitus: Are We There Yet?

Current nonpharmacological approaches, including diet and exercise interventions, for preventing and treating gestational diabetes mellitus are effective for less than 50% of women. Recent evidence suggests that the gut microbiome is integrally involved in maternal glucose homeostasis. Changes to the composition and metabolic behavior of the gut microbiota may play a role in the development and persistence of gestational diabetes mellitus. Thus, there is growing interest in targeting the maternal gut microbiome for preventing and managing pregnancy-related diseases including gestational diabetes mellitus. Future progress may come from a systems biology approach to elucidate the role of the gut microbiota in maternal glucose homeostasis.

Ligands, Receptors, and Transcription Factors that Mediate Inter-Cellular and Intra-Cellular Communication during Ovarian Follicle Development.

Reliably producing a competent oocyte entails a deeper comprehension of ovarian follicle maturation, a very complex process that includes meiotic maturation of the female gamete, the oocyte, together with the mitotic divisions of the hormone-producing somatic cells. In this report, we investigate murine ovarian folliculogenesis in vivo using publicly available time-series microarrays from primordial to antral stage follicles. Manually curated protein interaction networks were employed to identify autocrine and paracrine signaling between the oocyte and the somatic cells (granulosa and theca cells) at multiple stages of follicle development. We established plausible protein-binding interactions between expressed genes that encode secreted factors and expressed genes that encode cellular receptors. Some computationally identified signaling interactions are well established, such as the paracrine signaling from the oocyte to the somatic cells through the oocyte-secreted growth factor Gdf9, while others are novel connections in term of ovarian folliculogenesis, such as the possible paracrine connection from somatic-secreted factor Ntn3 to the oocyte receptor Neo1. Additionally, we identified several of the likely transcription factors that might control the dynamic transcriptome during ovarian follicle development, noting that the YAP/TAZ signaling pathway is very active in vivo. This novel dynamic model of signaling and regulation can be employed to generate testable hypotheses regarding follicle development that could be validated experimentally, guiding the improvement of culture media to enhance in vitro ovarian follicle maturation and possibly novel therapeutic targets for reproductive diseases.

Improving Mental Health for the Mother-Infant Dyad by Nutrition and the Maternal Gut Microbiome.

Perinatal mood and anxiety disorders (PMAD) have significant negative impacts on mother and child, yet treatments are limited. Adequate nutrition during the perinatal period is essential to maternal and infant health, including maternal mental health and the child's neurologic and neuropsychiatric development. Nutrition holds promise to improve prevention and treatment of PMAD. The ability to manipulate the gut microbiota composition and structure through host nutrition and to harness the gut microbes for improved individualized nutrition may be an important new direction for prevention and treatment of PMAD, thus improving the mental health of mother and child.

The human microbiota is associated with cardiometabolic risk across the epidemiologic transition.

Oral and fecal microbial biomarkers have previously been associated with cardiometabolic (CM) risk, however, no comprehensive attempt has been made to explore this association in minority populations or across different geographic regions. We characterized gut- and oral-associated microbiota and CM risk in 655 participants of African-origin, aged 25-45, from Ghana, South Africa, Jamaica, and the United States (US). CM risk was classified using the CM risk cut-points for elevated waist circumference, elevated blood pressure and elevated fasted blood glucose, low high-density lipoprotein (HDL), and elevated triglycerides. Gut-associated bacterial alpha diversity negatively correlated with elevated blood pressure and elevated fasted blood glucose. Similarly, gut bacterial beta diversity was also significantly differentiated by waist circumference, blood pressure, triglyceridemia and HDL-cholesterolemia. Notably, differences in inter- and intra-personal gut microbial diversity were geographic-region specific. Participants meeting the cut-points for 3 out of the 5 CM risk factors were significantly more enriched with Lachnospiraceae, and were significantly depleted of Clostridiaceae, Peptostreptococcaceae, and Prevotella. The predicted relative proportions of the genes involved in the pathways for lipopolysaccharides (LPS) and butyrate synthesis were also significantly differentiated by the CM risk phenotype, whereby genes involved in the butyrate synthesis via lysine, glutarate and 4-aminobutyrate/succinate pathways and LPS synthesis pathway were enriched in participants with greater CM risk. Furthermore, inter-individual oral microbiota diversity was also significantly associated with the CM risk factors, and oral-associated Streptococcus, Prevotella, and Veillonella were enriched in participants with 3 out of the 5 CM risk factors. We demonstrate that in a diverse cohort of African-origin adults, CM risk is significantly associated with reduced microbial diversity, and the enrichment of specific bacterial taxa and predicted functional traits in both gut and oral environments. As well as providing new insights into the associations between the gut and oral microbiota and CM risk, this study also highlights the potential for novel therapeutic discoveries which target the oral and gut microbiota in CM risk.

Decreased microbial co-occurrence network stability and SCFA receptor level correlates with obesity in African-origin women.

We compared the gut microbial populations in 100 women, from rural Ghana and urban US [50% lean (BMI < 25 kg/m2) and 50% obese (BMI ≥ 30 kg/m2)] to examine the ecological co-occurrence network topology of the gut microbiota as well as the relationship of short chain fatty acids (SCFAs) with obesity. Ghanaians consumed significantly more dietary fiber, had greater microbial alpha-diversity, different beta-diversity, and had a greater concentration of total fecal SCFAs (p-value < 0.002). Lean Ghanaians had significantly greater network density, connectivity and stability than either obese Ghanaians, or lean and obese US participants (false discovery rate (FDR) corrected p-value ≤ 0.01). Bacteroides uniformis was significantly more abundant in lean women, irrespective of country (FDR corrected p < 0.001), while lean Ghanaians had a significantly greater proportion of Ruminococcus callidus, Prevotella copri, and Escherichia coli, and smaller proportions of Lachnospiraceae, Bacteroides and Parabacteroides. Lean Ghanaians had a significantly greater abundance of predicted microbial genes that catalyzed the production of butyric acid via the fermentation of pyruvate or branched amino-acids, while obese Ghanaians and US women (irrespective of BMI) had a significantly greater abundance of predicted microbial genes that encoded for enzymes associated with the fermentation of amino-acids such as alanine, aspartate, lysine and glutamate. Similar to lean Ghanaian women, mice humanized with stool from the lean Ghanaian participant had a significantly lower abundance of family Lachnospiraceae and genus Bacteroides and Parabacteroides, and were resistant to obesity following 6-weeks of high fat feeding (p-value < 0.01). Obesity-resistant mice also showed increased intestinal transcriptional expression of the free fatty acid (Ffa) receptor Ffa2, in spite of similar fecal SCFAs concentrations. We demonstrate that the association between obesity resistance and increased predicted ecological connectivity and stability of the lean Ghanaian microbiota, as well as increased local SCFA receptor level, provides evidence of the importance of robust gut ecologic network in obesity.

Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells.

Breast cancer onset and disease progression have been linked to members of the TGFß superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGFß in malignancy, mediating both tumor suppressant and pro-metastatic behaviors. Overexpression of cell cycle regulators, cyclins D and E, renders cyclin-dependent kinases (CDKs) 4/2 hyperactive. Noncanonical phosphorylation of Smad3 by CDK4/2 inhibits tumor suppressant actions of Smad3. We hypothesized that CDK inhibition (CDKi) would restore Smad3 action and help promote cancer cell regression. Treatment of triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-436, Hs578T) with CDK2i or CDK4i resulted in increased Smad3 activity and decreased cell migration. Transfection with a 5M Smad3 construct containing inhibitory mutations in 5 CDK phosphorylation sites also resulted in decreased TNBC cell migration and invasion. MDA-MB-231 cells treated with CDK2i or CDK4i resulted in decreased Smad3 protein phosphorylation at the CDK phosphorylation T179 site, decreased MMP2 and c-myc expression, and increased p15 and p21 expression. Using a novel transfected cell array, we found that CDK2i treatment decreased activity of the epithelial-to-mesenchymal transition related transcription factors Snail and Twist. In vivo studies in an MDA-MB-231 tumor model showed that individual and combination treatment with paclitaxel and CDK2i resulted in decreased tumor volume and Ki67 staining. Collectively, these data support further investigation of targeted CDK inhibitors as a promising therapeutic strategy for TNBC, a breast cancer subtype with limited treatment options.

Promoting extracellular matrix remodeling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels.

The in vitro growth of ovarian follicles is an emerging technology for fertility preservation. Various strategies support the culture of secondary and multilayer follicles from various species including mice, non-human primate, and human; however, the culture of early stage (primary and primordial) follicles, which are more abundant in the ovary and survive cryopreservation, has been limited. Hydrogel-encapsulating follicle culture systems that employed feeder cells, such as mouse embryonic fibroblasts (MEFs), stimulated the growth of primary follicles (70-80 µm); yet, survival was low and smaller follicles (<70 µm) rapidly lost structure and degenerated. These morphologic changes were associated with a breakdown of the follicular basement membrane; hence, this study investigated ascorbic acid based on its role in extracellular matrix (ECM) deposition/remodeling for other applications. The selection of ascorbic acid was further supported by a microarray analysis that suggested a decrease in mRNA levels of enzymes within the ascorbate pathway between primordial, primary, and secondary follicles. The supplementation of ascorbic acid (50 µg/mL) significantly enhanced the survival of primary follicles (<80 µm) cultured in alginate hydrogels, which coincided with improved structural integrity. Follicles developed antral cavities and increased to diameters exceeding 250 µm. Consistent with improved structural integrity, the gene/protein expression of ECM and cell adhesion molecules was significantly changed. This research supports the notion that modifying the culture environment (medium components) can substantially enhance the survival and growth of early stage follicles.

Dynamic transcription factor activity profiling in 2D and 3D cell cultures.

Live-cell assays to measure cellular function performed within 3D cultures have the potential to elucidate the underlying processes behind disease progression and tissue formation. Cells cultured in 3D interact and remodel their microenvironment and can develop into complex structures. We have developed a transcription factor (TF) activity array that uses bioluminescence imaging (BLI) of lentiviral delivered luminescent reporter constructs that allows for the non-invasive imaging of TF activity in both 2D and 3D culture. Imaging can be applied repeatedly throughout culture to capture dynamic TF activity, though appropriate normalization is necessary. We investigated in-well normalization using Gaussia or Renilla luciferase, and external well normalization using firefly luciferase. Gaussia and Renilla luciferase were each unable to provide consistent normalization for long-term measurement of TF activity. However, external well normalization provided low variability and accounted for changes in cellular dynamics. Using external normalization, dynamic TF activities were quantified for five TFs. The array captured expected changes in TF activity to stimuli, however the array also provided dynamic profiles within 2D and 3D that have not been previously characterized. The development of the technology to dynamically track TF activity within cells cultured in both 2D and 3D can provide greater understanding of complex cellular processes.

Microarray analysis identifies COMP as the most differentially regulated transcript throughout in vitro follicle growth.

In vitro follicle growth has emerged as a technology that can provide new information about folliculogenesis and serve as part of a suite of methods currently under development to assist women whose fertility is threatened by cancer treatments. Though it has been shown that in vitro-grown follicles secrete peptide and steroid hormones, much of the follicular transcriptome remains unknown. Thus, microarray analysis was performed to characterize the transcriptome and secretome of in vitro-grown follicles. One prominently regulated gene product was cartilage oligomeric matrix protein (Comp): its mRNA was upregulated during the final 4 days of culture (P < 0.05) and COMP protein could be detected in medium from individual follicles. COMP expression localized to mural granulosa cells of large antral follicles both in vitro and in vivo, with maximal expression immediately preceding ovulation in cycling and chorionic gonadotropin-primed female mice. COMP was co-expressed with two known markers of follicle maturation, inhibin ß(A) and gremlin, and was expressed only in TUNEL-negative follicles. In addition to other gene products identified in the microarray, COMP has potential utility as a marker of follicle maturation.

State-time spectrum of signal transduction logic models.

Despite the current wealth of high-throughput data, our understanding of signal transduction is still incomplete. Mathematical modeling can be a tool to gain an insight into such processes. Detailed biochemical modeling provides deep understanding, but does not scale well above relatively a few proteins. In contrast, logic modeling can be used where the biochemical knowledge of the system is sparse and, because it is parameter free (or, at most, uses relatively a few parameters), it scales well to large networks that can be derived by manual curation or retrieved from public databases. Here, we present an overview of logic modeling formalisms in the context of training logic models to data, and specifically the different approaches to modeling qualitative to quantitative data (state) and dynamics (time) of signal transduction. We use a toy model of signal transduction to illustrate how different logic formalisms (Boolean, fuzzy logic and differential equations) treat state and time. Different formalisms allow for different features of the data to be captured, at the cost of extra requirements in terms of computational power and data quality and quantity. Through this demonstration, the assumptions behind each formalism are discussed, as well as their advantages and disadvantages and possible future developments.

The impact of adhesion peptides within hydrogels on the phenotype and signaling of normal and cancerous mammary epithelial cells.

The microenviroment contributes to directing mammary epithelial cell (MEC) development and the progression of breast cancer. Three-dimensional culture models have been used to support formation of structures that display varying degrees of disorganization that parallel the degree of cancer. Synthetic hydrogels were employed to investigate the mechanisms by which specific adhesion signals in the microenvironment directed development. Polyethylene glycol-based hydrogels supported 3D growth of MECs and directed formation of a range of phenotypes that were functions of genotype, and identity and concentration of adhesion peptides RGD and YIGSR. Non-cancerous and cancerous MECs responded differentially to the same adhesion cues and produced variable structural organizations. An analysis of dynamic signaling pathways revealed differential activities of transcription factors within the MAPK and JAK/STAT pathways in response to genotype and adhesion. These results directly implicate adhesion in cancer development and demonstrate that AP1, CREB, STAT1, and STAT3 all contribute to the genotype dependence of cellular response to adhesion peptides. The tools presented in this work could be applied to other systems and connect extracellular cues with intracellular signaling to molecularly dissect tissue development and further biomaterials development.