Nicotinic acetylcholine receptor CHRNA5 is overexpressed in head and neck squamous cell carcinoma patients with a recent tobacco smoking history.

Tobacco smoking is a major driver of head and neck squamous cell carcinoma (HNSCC) occurrence, and previous studies have shed light on the precise molecular alterations in tobacco-related HNSCCs when compared to HNSCCs associated with other risk factors (ex: human papillomavirus/HPV status). In this study, we analyzed the gene expression differences in HNSCC cases with a recent smoking history and revealed that the nicotinic acetylcholine receptor CHRNA5 is differentially overexpressed in smoking-related HNSCCs. CHRNA5 overexpression in these HNSCCs corresponds with a worse prognosis and is inversely correlated with an immune expression signature commonly associated with better prognosis. From these results, our study highlights the potential role of the nicotine-activated CHRNA5 receptor in HNSCC progression and corresponds with other recent reports highlighting the potential role of nicotine induction in promoting cancer progression.

The physical-mental health interface in the preconception period: analysis of 131 182 women planning pregnancy in the UK.

OBJECTIVE: The physical and mental health of women prior to conception can have a significant impact on pregnancy and child outcomes. Given the rising burden of non-communicable diseases, the aim was to explore the relation between mental health, physical health and health behaviour in women planning a pregnancy. METHODS: Cross-sectional analysis of responses from 131 182 women to a preconception health digital education tool, providing data on physical and mental health and health behaviour. Logistic regression was used to explore associations between mental health and physical health variables. RESULTS: Physical health conditions were reported by 13.1% and mental health conditions by 17.8%. There was evidence for an association between self-reported physical and mental health conditions (odds ratio [OR] 2.22, 95% CI 2.14-2.3). Those with a mental health condition were less likely to engage with healthy behaviour at preconception such as folate supplementation (OR 0.89, 95% CI 0.86-0.92) and consumption of the recommended amount of fruit and vegetables (OR 0.77, 95% CI 0.74-0.79). They were more likely to be physically inactive (OR 1.14, 95% CI 1.11-1.18), smoke tobacco (OR 1.72, 95% CI 1.66-1.78) and use illicit substances (OR 2.4, 95% CI 2.25-2.55). CONCLUSIONS: Greater recognition of mental and physical comorbidities is needed and closer integration of physical and mental healthcare in the preconception period, which could support people to optimise their health during this time and improve long-term outcomes.

Pre-pregnancy health of women with pre-existing diabetes or previous gestational diabetes: Analysis of pregnancy risk factors and behavioural data from a digital tool.

AIMS: To examine health behaviours and risk factors in women with pre-existing diabetes or previous gestational diabetes mellitus who are planning pregnancy. METHODS: Health behaviour, risk factor and demographic data obtained from a digital pregnancy planning advisory tool (Tommy's charity UK) were analysed. Descriptive statistical analysis was performed, stratified by diabetes type. RESULTS: Data from 84,359 women, including 668 with type 1 diabetes, 707 with type 2 diabetes and 1785 with previous gestational diabetes obtained over a 12-month period (September 2019-September 2020) were analysed. 65%, 95%CI (61,68%) of women with type 2 diabetes and 46%, 95%CI (43,48%) with previous gestational diabetes were obese (BMI ≥30 kg/m2 ), compared with 26%, 95%CI (26,26%) without diabetes. Use of folic acid supplements was low; 41%, 95%CI (40,41%) of women without diabetes and 42%, 95%CI (40,45%) with previous gestational diabetes reported taking folic acid (any dose) while 47%, 95%CI (43.50%) women with type 1 diabetes and 44%, 95%CI (40,47%) women with type 2 diabetes respectively reported taking the recommended dose (5 mg). More women with type 1 diabetes and type 2 diabetes reported smoking (20%, 95%CI [17,23%] and 23%, 95%CI [20,26%] respectively) and taking illicit/recreational drugs (7%, 95%CI [6,10%] and 9%, 95% CI [7,11%]) compared to women without diabetes (smoking 17%, 95% CI [16,17%], drug use 5%, 95%CI [5,5%]). Alcohol consumption, low levels of physical activity and of fruit and vegetable intake were also evident. CONCLUSIONS: This study highlights the potential of online pregnancy planning advisory tools to reach high-risk women and emphasises the need to improve pre-pregnancy care for women with pre-existing diabetes and previous gestational diabetes, many of whom are actively seeking advice. It is also the first to describe pre-pregnancy health behaviours in women with previous gestational diabetes.

Community Pharmacist-Led Interventions to Improve Preconception and Pregnancy Health: A Systematic Review.

Background: Community pharmacist-led interventions are effective in improving health outcomes; however, their impact in improving preconception and pregnancy health is not clear. This study evaluated the effectiveness of community pharmacist-led interventions which aimed to improve health outcomes of preconception and pregnant women. Methods: A systematic review of the literature, consistent with PRISMA guidelines, was performed. Five electronic databases were searched up to February 2021. Results: Four studies, three in pregnant women and one in preconception women, were identified. The studies focused on improving micronutrient status and smoking cessation. The studies increased knowledge about, and use of, iron supplements, and improved iron status and smoking cessation rates in pregnant women, while improving knowledge regarding, and increasing the use of, preconception folic acid. The studies were ranked as weak to moderate quality. Conclusion: This review provides preliminary evidence for the potential benefit of community pharmacist-led interventions to improve the health of women before and during pregnancy.

Health behaviours in 131,182 UK women planning pregnancy.

BACKGROUND: A woman's health at the time of conception lays the foundation for a healthy pregnancy and the lifelong health of her child. We investigated the health behaviours of UK women planning pregnancy. METHODS: We analysed survey data from the 'Planning for Pregnancy' online tool (Tommy's, UK). We described all women planning pregnancy and compared the frequency of non-adherence to preconception recommendations in women who had already stopped contraception (active planners) and those who had not (non-active planners). RESULTS: One hundred thirty-one thousand one hundred eighty-two women from across the UK were included, of whom 64.8% were actively planning pregnancy. Of the whole cohort, twenty percent were smokers and less than one third took folic acid supplements (31.5%). Forty two percent engaged in less than the recommended 150 min of weekly physical activity and only 53.3% consumed five portions of fruit or vegetables 4 days a week. Smokers were 1.87 times more likely to be active planners than non-smokers (95% CI 1.79-1.94), and women who took folic acid were 7 times more likely to be active planners (95% CI 6.97-7.59) compared to women who did not. Smoking, drug use and lack of folic acid supplementation were common in younger women and those who were underweight. CONCLUSIONS: This unique survey of UK women has identified poor adherence to preconception recommendations in those planning pregnancies and supports the need for a greater public health focus on preconception health. This study provides a contemporary basis from which to inform preconception health advice and a benchmark to measure changes over time.

A preconception intervention targeted at women with modifiable risk factors before pregnancy to improve outcomes; protocol for the Get Ready! feasibility trial.

BACKGROUND: The health of a woman before conception not only influences the outcome of her pregnancy but also the lifelong health of mother and child. Many women in the UK are inadequately prepared for pregnancy, with reports of a high prevalence of smoking, low folic acid supplement use, and suboptimal diet and physical activity. Get Ready! will link an online digital tool to identify women planning pregnancy most at risk of complications with a personalised intervention to improve health behaviours and biomarkers of metabolic health. METHODS: Women planning pregnancy will be identified from a free and widely used online preconception tool. A short online screening questionnaire will then be used to recruit women considered to be at high metabolic risk. Eligibility criteria include resident in the UK, age > 18-< 50 years, BMI ≥ 23 kg/m2 (South Asian) or ≥ 25 kg/m2 (all other ethnicities), and plus one or more of the following: 1st degree relative with type 2 diabetes, previous gestational diabetes (GDM), previous baby > 4 kg, or high risk ethnicity for GDM. Eligible women who consent to participate will be enrolled in a commercially available preconception intervention (Prepare Plans, LiveSmart UK Ltd). Following an online health assessment and home blood test, women will be provided with individualised lifestyle advice and coaching by dietitians. Process evaluation will provide an assessment of implementation of the intervention. Change in health behaviours and biomarkers of metabolic health will also be examined. DISCUSSION: Suboptimal health behaviours amongst women planning pregnancy are widely prevalent in the UK. Personalised health checks and coaching are especially important for women at risk of pregnancy complications. Get Ready! introduces a novel approach to identifying high risk women planning pregnancy and provision of a targeted intervention. REGISTRATION: Trial sponsor: King's College London.

Maternal dysglycaemia, changes in the infant's epigenome modified with a diet and physical activity intervention in pregnancy: Secondary analysis of a randomised control trial.

BACKGROUND: Higher maternal plasma glucose (PG) concentrations, even below gestational diabetes mellitus (GDM) thresholds, are associated with adverse offspring outcomes, with DNA methylation proposed as a mediating mechanism. Here, we examined the relationships between maternal dysglycaemia at 24 to 28 weeks' gestation and DNA methylation in neonates and whether a dietary and physical activity intervention in pregnant women with obesity modified the methylation signatures associated with maternal dysglycaemia. METHODS AND FINDINGS: We investigated 557 women, recruited between 2009 and 2014 from the UK Pregnancies Better Eating and Activity Trial (UPBEAT), a randomised controlled trial (RCT), of a lifestyle intervention (low glycaemic index (GI) diet plus physical activity) in pregnant women with obesity (294 contol, 263 intervention). Between 27 and 28 weeks of pregnancy, participants had an oral glucose (75 g) tolerance test (OGTT), and GDM diagnosis was based on diagnostic criteria recommended by the International Association of Diabetes and Pregnancy Study Groups (IADPSG), with 159 women having a diagnosis of GDM. Cord blood DNA samples from the infants were interrogated for genome-wide DNA methylation levels using the Infinium Human MethylationEPIC BeadChip array. Robust regression was carried out, adjusting for maternal age, smoking, parity, ethnicity, neonate sex, and predicted cell-type composition. Maternal GDM, fasting glucose, 1-h, and 2-h glucose concentrations following an OGTT were associated with 242, 1, 592, and 17 differentially methylated cytosine-phosphate-guanine (dmCpG) sites (false discovery rate (FDR) ≤ 0.05), respectively, in the infant's cord blood DNA. The most significantly GDM-associated CpG was cg03566881 located within the leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) (FDR = 0.0002). Moreover, we show that the GDM and 1-h glucose-associated methylation signatures in the cord blood of the infant appeared to be attenuated by the dietary and physical activity intervention during pregnancy; in the intervention arm, there were no GDM and two 1-h glucose-associated dmCpGs, whereas in the standard care arm, there were 41 GDM and 160 1-h glucose-associated dmCpGs. A total of 87% of the GDM and 77% of the 1-h glucose-associated dmCpGs had smaller effect sizes in the intervention compared to the standard care arm; the adjusted r2 for the association of LGR6 cg03566881 with GDM was 0.317 (95% confidence interval (CI) 0.012, 0.022) in the standard care and 0.240 (95% CI 0.001, 0.015) in the intervention arm. Limitations included measurement of DNA methylation in cord blood, where the functional significance of such changes are unclear, and because of the strong collinearity between treatment modality and severity of hyperglycaemia, we cannot exclude that treatment-related differences are potential confounders. CONCLUSIONS: Maternal dysglycaemia was associated with significant changes in the epigenome of the infants. Moreover, we found that the epigenetic impact of a dysglycaemic prenatal maternal environment appeared to be modified by a lifestyle intervention in pregnancy. Further research will be needed to investigate possible medical implications of the findings. TRIAL REGISTRATION: ISRCTN89971375.

Do nuclear magnetic resonance (NMR)-based metabolomics improve the prediction of pregnancy-related disorders? Findings from a UK birth cohort with independent validation.

BACKGROUND: Prediction of pregnancy-related disorders is usually done based on established and easily measured risk factors. Recent advances in metabolomics may provide earlier and more accurate prediction of women at risk of pregnancy-related disorders. METHODS: We used data collected from women in the Born in Bradford (BiB; n = 8212) and UK Pregnancies Better Eating and Activity Trial (UPBEAT; n = 859) studies to create and validate prediction models for pregnancy-related disorders. These were gestational diabetes mellitus (GDM), hypertensive disorders of pregnancy (HDP), small for gestational age (SGA), large for gestational age (LGA) and preterm birth (PTB). We used ten-fold cross-validation and penalised regression to create prediction models. We compared the predictive performance of (1) risk factors (maternal age, pregnancy smoking, body mass index (BMI), ethnicity and parity) to (2) nuclear magnetic resonance-derived metabolites (N = 156 quantified metabolites, collected at 24-28 weeks gestation) and (3) combined risk factors and metabolites. The multi-ethnic BiB cohort was used for training and testing the models, with independent validation conducted in UPBEAT, a multi-ethnic study of obese pregnant women. RESULTS: Maternal age, pregnancy smoking, BMI, ethnicity and parity were retained in the combined risk factor and metabolite models for all outcomes apart from PTB, which did not include maternal age. In addition, 147, 33, 96, 51 and 14 of the 156 metabolite traits were retained in the combined risk factor and metabolite model for GDM, HDP, SGA, LGA and PTB, respectively. These include cholesterol and triglycerides in very low-density lipoproteins (VLDL) in the models predicting GDM, HDP, SGA and LGA, and monounsaturated fatty acids (MUFA), ratios of MUFA to omega 3 fatty acids and total fatty acids, and a ratio of apolipoprotein B to apolipoprotein A-1 (APOA:APOB1) were retained predictors for GDM and LGA. In BiB, discrimination for GDM, HDP, LGA and SGA was improved in the combined risk factors and metabolites models. Risk factor area under the curve (AUC 95% confidence interval (CI)): GDM (0.69 (0.64, 0.73)), HDP (0.74 (0.70, 0.78)) and LGA (0.71 (0.66, 0.75)), and SGA (0.59 (0.56, 0.63)). Combined risk factor and metabolite models AUC 95% (CI): GDM (0.78 (0.74, 0.81)), HDP (0.76 (0.73, 0.79)) and LGA (0.75 (0.70, 0.79)), and SGA (0.66 (0.63, 0.70)). For GDM, HDP and LGA, but not SGA, calibration was good for a combined risk factor and metabolite model. Prediction of PTB was poor for all models. Independent validation in UPBEAT at 24-28 weeks and 15-18 weeks gestation confirmed similar patterns of results, but AUCs were attenuated. CONCLUSIONS: Our results suggest a combined risk factor and metabolite model improves prediction of GDM, HDP and LGA, and SGA, when compared to risk factors alone. They also highlight the difficulty of predicting PTB, with all models performing poorly.

The UK Pregnancies Better Eating and Activity Trial (UPBEAT); Pregnancy Outcomes and Health Behaviours by Obesity Class.

The effectiveness of antenatal intervention in women with increasing obesity is unknown. This study investigated whether there was a differential effect of antenatal intervention on diet, physical activity and pregnancy outcomes in women stratified by obesity class using data from the UK Pregnancies Better Eating and Activity Trial (UPBEAT) (n = 1555). The stratification was by World Health Organization classifications: Class I, II and III (30-34.9 kg/m2, 35-39.9 kg/m2 and ≥40 kg/m2). Using linear and logistic regression, adjusted for confounders, outcomes were assessed post-intervention (27+0-28+6 weeks' gestation) and in late pregnancy (34+0-36+0 weeks' gestation). Interactions between obesity class and the intervention were explored. Compared to the standard care arm, class III intervention women had lower gestational weight gain (GWG) (-1.87 kg; 95% CI -3.29 to -0.47, p = 0.009), and the effect of the intervention was greater in class III compared to class I, by -2.01 kg (95% CI -3.45 to -0.57, p = 0.006). Class I and II intervention women reported significantly lower dietary glycaemic load and saturated fat intake across their pregnancy. This differential effect of the intervention suggests antenatal interventions for women with obesity should stratify outcomes by obesity severity. This would inform evidence-based antenatal strategies for high-risk groups, including women with a BMI ≥ 40 kg/m2.

Influence of GDM Diagnosis and Treatment on Weight Gain, Dietary Intake and Physical Activity in Pregnant Women with Obesity: Secondary Analysis of the UPBEAT Study.

Obesity during pregnancy is associated with the development of gestational diabetes (GDM). This study aimed to assess if the result of an oral glucose tolerance test (OGTT) for GDM influences health (diet and physical activity) behaviours of pregnant women with obesity. In total, 1031 women who participated in the UK Pregnancies Better Eating and Activity Trial (UPBEAT) of a lifestyle intervention from early pregnancy were included. Changes in weight gain, dietary intake and physical activity following an OGTT undertaken between 27+0 and 28+6 weeks' and 34 and 36 weeks' gestation were examined using linear regression with appropriate adjustment for confounders. Obese women without GDM (IADPSG criteria) gained 1.9 kg (95% CI -2.2, -1.5, p < 0.001) more weight than women with GDM. Women with GDM demonstrated greater reductions in energy (-142kcal, 95%CI -242.2, -41.9, p = 0.006), carbohydrate intake (-1.5%E 95%CI -2.8, -0.3, p = 0.016) and glycaemic load (-15.2, 95%CI -23.6, -6.7, p < 0.001) and a greater increase in protein intake (2%E, 95%CI 1.3, 2.7, p < 0.001), compared to women without GDM. Trial intervention allocation did not influence any associations observed. The findings emphasise the need for strategies to optimise the health behaviours of pregnant women with obesity, following a negative OGTT for GDM.

Smartphone applications available to pregnant women in the United Kingdom: An assessment of nutritional information.

The importance of diet during pregnancy is critically important for the short- and long-term health of both mother and child. The number of apps targeting pregnant women is rapidly increasing, yet the nutritional content of these tools remains largely unexplored. This review aimed to evaluate the coverage and content of nutrition information in smartphone apps available to U.K. pregnant women. Keyword searches were conducted in iTunes and Google Play stores in November 2018. Candidate apps were included if they targeted pregnant women, provided pregnancy-specific nutritional information, had a user rating of at least 4+ based on at least 20 ratings, and were available in English. Nutritional content was assessed for accuracy against U.K. recommendations. Behaviour change techniques (BCTs) were also evaluated. Twenty-nine apps were included, seven of which originated in the United Kingdom. There was a large variability in the quality of smartphone app nutritional information. The accuracy of nutrition information varied, and several apps conveyed inappropriate information for pregnancy. On average, 10 BCTs were identified per app (range 2-15). Overall, smartphone apps do not consistently provide accurate and useful nutritional information to pregnant women. This study highlights the need for the integration of evidence-based nutritional information during app development and for increased regulatory oversight. App developers should also make it clear that nutritional content is intended for a specific geographical region or population or modify for the intended audience. These are important considerations for the design of future apps, which are increasingly used to complement existing maternity services.

Relationships between Maternal Obesity and Maternal and Neonatal Iron Status.

: Obesity in pregnancy may negatively influence maternal and infant iron status. The aim of this study was to examine the association of obesity with inflammatory and iron status in both mother and infant in two prospective studies in pregnancy: UPBEAT and SCOPE. Maternal blood samples from obese (n = 245, BMI ≥ 30 kg/m²) and normal weight (n = 245, BMI < 25 kg/m²) age matched pregnant women collected at approximately 15 weeks' gestation, and umbilical cord blood samples collected at delivery, were analysed for a range of inflammatory and iron status biomarkers. Concentrations of C- reactive protein and Interleukin-6 in obese women compared to normal weight women were indicative of an inflammatory response. Soluble transferrin receptor (sTfR) concentration [18.37 nmol/L (SD 5.65) vs 13.15 nmol/L (SD 2.33)] and the ratio of sTfR and serum ferritin [1.03 (SD 0.56) vs 0.69 (SD 0.23)] were significantly higher in obese women compared to normal weight women (P < 0.001). Women from ethnic minority groups (n = 64) had higher sTfR concentration compared with white women. There was no difference in maternal hepcidin between obese and normal weight women. Iron status determined by cord ferritin was not statistically different in neonates born to obese women compared with neonates born to normal weight women when adjusted for potential confounding variables. Obesity is negatively associated with markers of maternal iron status, with ethnic minority women having poorer iron statuses than white women.

Outcomes of Cancer Survivorship Care Provided by Advanced Practice RNs Compared to Other Models of Care: A Systematic Review

Problem Identification: Advanced practice RNs (APRNs) have become leaders in providing survivorship care. However, outcomes of survivorship care provided by APRNs compared to other providers remain unclear. Literature Search: A systematic literature search included articles published after 2005 that described a survivorship model of care and use of a survivorship care plan (SCP), as well as reported outcomes. Data Evaluation: Sixteen studies were appraised and ranked by strength. Literature was evaluated based on the model of care, which included physicians, nurses, and APRNs who provided or reviewed SCPs. Outcomes evaluated were satisfaction, quality of life (QOL), and process/cost efficiency. Synthesis: Survivorship care is reimbursable when provided by APRNs secondary to the ability to bill for services. Improved patient satisfaction and QOL are demonstrated when survivorship care is provided by APRNs. Incorporation of SCPs into health records improves process/cost efficiency. Conclusions: Patient satisfaction was reported in all models of care. When compared to groups who received no survivorship care, no differences were reported in QOL, but survivorship care required extensive use of resources. Survivorship care provided by APRNs demonstrated improvement in satisfaction, QOL, and process/cost efficiency. Implications for Nursing: Incorporating descriptions of care models and associated outcomes into randomized, controlled trials of survivorship care would provide stronger evidence to guide practice. Studies evaluating outcomes of process/cost efficiency should be considered for future research. Outcome research is needed regarding the incorporation of SCPs into electronic health records.

Prevalence and Risk Factors Associated with Musculoskeletal Discomfort in Spay and Neuter Veterinarians.

A cross-sectional study to investigate musculoskeletal discomfort (MSD) surveyed 219 veterinarians who currently or previously perform spays and neuters at least 4 hours per week. Participants were asked about the presence and severity of hand and body MSD during the previous month, whether MSD interfered with work or daily activities, whether they attributed their MSD to their spay/neuter work, and whether MSD had ever necessitated absence from work. The period prevalence of MSD was 99.1%, with 76.7% experiencing hand or wrist pain and 98.2% experiencing body pain. Hand discomfort was most commonly reported in the right thumb and/or thumb base (49.8%) and the right wrist (37.9%). Body discomfort was most commonly reported in the lower back (76.7%), shoulders (72.6%), and neck (71.7%). Increasing career length, increasing weekly hours in surgery and decreasing job satisfaction were the work-related factors with the greatest relative contribution accounting for variation in hand pain severity and total pain. Although 94.4% of respondents felt that posture during surgery is important, only 30.6% had received any instruction in posture and positioning for surgery. Future interventions should aim to optimize surgical efficiency, surgeon work schedules, and working environment. Analysis and intervention studies are required to determine further causes of MSD in these veterinarians and develop interventions to prevent MSD.

Formation of the digestive system in zebrafish: III. Intestinal epithelium morphogenesis.

Recent analysis of a novel strain of transgenic zebrafish (gutGFP) has provided a detailed description of the early morphological events that occur during the development of the liver and pancreas. In this paper, we aim to complement these studies by providing an analysis of the morphological events that shape the zebrafish intestinal epithelium. One of our goals is to provide a framework for the future characterization of zebrafish mutant phenotypes in which intestinal epithelial morphogenesis has been disrupted. Our analysis encompasses the period between 26 and 126 h post-fertilization (hpf) and follows the growth, lumen formation and differentiation of a continuous layer of endoderm into a functional intestinal epithelium with three morphologically distinct segments: the intestinal bulb, mid-intestine and posterior intestine. Between 26 hpf and 76 hpf, the entire intestinal endoderm is a highly proliferative organ. To make a lumen, the zebrafish endoderm cells undergo apical membrane biogenesis, adopt a bilayer configuration and form small cavities that coalesce without cell death. Thereafter, the endoderm cells polarize and differentiate into distinct cell lineages. Enteroendocrine cells are distinguished first at 52 hpf in the caudal region of the intestine in a new stable transgenic line, Tg[nkx2.2a:mEGFP]. The differentiation of mucin-containing goblet cells is first evident at 100 hpf and is tightly restricted to a middle segment of the intestine, designated the mid-intestine, that is also demarcated by the presence of enterocytes with large supranuclear vacuoles. Meanwhile, striking expansion of the lumen in the rostral intestine forms the intestinal bulb. Here the epithelium elaborates folds and proliferating cells become progressively restricted to a basal compartment analogous to the crypts of Lieberkühn in mammals. At 126 hpf, the posterior intestine remains an unfolded monolayer of simple columnar epithelium.

The role of disulfide bonds in the structure and function of murine epidermal growth factor (mEGF).

A systematic study using solid phase peptide synthesis has been undertaken to examine the role of the disulfide bonds in the structure and function of mEGF. A combination of one, two and three native disulfide pair analogues of an active truncated (4-48) form of mEGF have been synthesised by replacing specific cysteine residues with isosteric a-amino-n-butyric acid (Abu). Oxidation of the peptides was performed using either conventional aerobic oxidation at basic pH, in DMSO under acidic conditions or via selective disulfide formation using orthogonal protection of the cysteine pairs. The contribution of individual, or pairs of, disulfide bonds to EGF structure was evaluated by CD and (1)H-NMR spectroscopy. The mitogenic activity of each analogue was determined using Balb/c 3T3 mouse fibroblastsAs we have reported previously (Barnham et al. 1998), the disulfide bond between residues 6 and 20 can be removed with significant retention of biological activity (EC50 20-50 nM). The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. We now show that removal of any other disulfide bond, either singly or in pairs, results in a major disruption of the tertiary structure, and a large loss of activity (EC50>900 nM). Remarkably, the linear analogue appears to have greater activity (EC50 580 nM) than most one and two disulfide bond analogues although it does not have a definable tertiary structure.

Analysis of the regulation of the A33 antigen gene reveals intestine-specific mechanisms of gene expression.

The A33 antigen is a transmembrane protein expressed almost exclusively by intestinal epithelial cells. The level of its expression is robust and uniform throughout the rostrocaudal axis of the human and mouse intestines. In the colon, strong expression is found in the basolateral membranes of both the proliferating cells in the lower regions of the crypts and the differentiating cells in the upper regions of crypts. Similarly, in the small intestine, the protein is highly expressed by all the epithelial cells in the crypts and by the differentiated cells migrating over the villi. Thus, the A33 antigen has emerged as a definitive marker for all intestinal epithelial cells, irrespective of cell lineage and differentiation status. To understand the molecular mechanisms mediating this rare tissue-specific expression pattern, we undertook a comprehensive analysis of the 5'-regulatory region of the human A33 antigen gene. This allowed us to point to positive cis-regulatory elements incorporating consensus Krüppel-like factor and caudal-related homeobox (CDX)-binding sites, located just upstream from the human A33 antigen transcription start site, as being important for the intestine-specific expression pattern of this gene. Further analysis provided evidence that the A33 antigen gene may be one of only a few target genes to be described thus far for the intestine-specific homeobox transcription factor, CDX1. Taken together, our data lead us to propose that the activity of CDX1 is pivotal in mediating the exquisite, intestine-specific expression pattern of the A33 antigen gene.