Protein Micropatterning in 2.5D: An Approach to Investigate Cellular Responses in Multi-Cue Environments.

The extracellular microenvironment is an important regulator of cell functions. Numerous structural cues present in the cellular microenvironment, such as ligand distribution and substrate topography, have been shown to influence cell behavior. However, the roles of these cues are often studied individually using simplified, single-cue platforms that lack the complexity of the three-dimensional, multi-cue environment cells encounter in vivo. Developing ways to bridge this gap, while still allowing mechanistic investigation into the cellular response, represents a critical step to advance the field. Here, we present a new approach to address this need by combining optics-based protein patterning and lithography-based substrate microfabrication, which enables high-throughput investigation of complex cellular environments. Using a contactless and maskless UV-projection system, we created patterns of extracellular proteins (resembling contact-guidance cues) on a two-and-a-half-dimensional (2.5D) cell culture chip containing a library of well-defined microstructures (resembling topographical cues). As a first step, we optimized experimental parameters of the patterning protocol for the patterning of protein matrixes on planar and non-planar (2.5D cell culture chip) substrates and tested the technique with adherent cells (human bone marrow stromal cells). Next, we fine-tuned protein incubation conditions for two different vascular-derived human cell types (myofibroblasts and umbilical vein endothelial cells) and quantified the orientation response of these cells on the 2.5D, physiologically relevant multi-cue environments. On concave, patterned structures (curvatures between κ = 1/2500 and κ = 1/125 µm-1), both cell types predominantly oriented in the direction of the contact-guidance pattern. In contrast, for human myofibroblasts on micropatterned convex substrates with higher curvatures (κ ≥ 1/1000 µm-1), the majority of cells aligned along the longitudinal direction of the 2.5D features, indicating that these cells followed the structural cues from the substrate curvature instead. These findings exemplify the potential of this approach for systematic investigation of cellular responses to multiple microenvironmental cues.

Supramolecular Additive-Initiated Controlled Atom Transfer Radical Polymerization of Zwitterionic Polymers on Ureido-pyrimidinone-Based Biomaterial Surfaces.

Surface-initiated controlled radical polymerization is a popular technique for the modification of biomaterials with, for example, antifouling polymers. Here, we report on the functionalization of a supramolecular biomaterial with zwitterionic poly(sulfobetaine methacrylate) via atom transfer radical polymerization from a macroinitiator additive, which is embedded in the hard phase of the ureido-pyrimidinone-based material. Poly(sulfobetaine methacrylate) was successfully polymerized from these surfaces, and the polymerized sulfobetaine content, with corresponding antifouling properties, depended on both the macroinitiator additive concentration and polymerization time. Furthermore, the polymerization from the macroinitiator additive was successfully translated to functional electrospun scaffolds, showing the potential for this functionalization strategy in supramolecular material systems.

A new species of African mole-rat (Fukomys, Bathyergidae, Rodentia) from the Zaire-Zambezi watershed.

A new species of bathyergid mole-rat, Fukomys vandewoestijneae, is described from an area on the Zaïre-Zambezi watershed, centred on the Ikelenge pedicle in the North-Western province of Zambia. It is diagnosed by a unique combination of morphological (size, lack of clear headmarks), chromosomal (2n= 44) and DNA sequence characteristics. This medium-sized species belongs to the Giant mole-rat "F. mechowii" clade, which was hitherto considered monotypic. Its known distribution is limited to the Ikelenge pedicle of Zambia and adjacent areas in the Democratic Republic of Congo (DRC) and presumably Angola. Colonies of this social mole-rat were observed in the chanas (dambos), degraded miombo woodland and in villages. Although presumably sympatric in historical times with F. inechowii, no overlap in the species current distribution could be established. This local endemic species adds further evidence to the conservation importance of the two-pedicle region (Ikelenge pedicle (Zambia-Katanga pedicle (DRC)).

A two-state model for Ca2+/CaM-dependent protein kinase II (alphaCaMKII) in response to persistent Ca2+ stimulation in hippocampal neurons.

Persistent elevation of the intracellular free Ca(2+) concentration [Ca(2+)](i) is neurotoxic and therefore it is important to understand how it affects downstream components of the Ca(2+) signaling pathway. The response of calmodulin (CaM) and alphaCa(2+)/CaM-dependent protein kinase II (alphaCaMKII), to intracellular Ca(2+) overload in hippocampal neurons is studied by confocal imaging of fluorescently tagged proteins. Transient and persistent redistribution of CaM and alphaCaMKII together is seen from the cytosol to dendritic and somatic punctae. Typical persistent redistribution occurs following a lag of 138+/-(S.E.M.) 12 s and is complete at 460+/-(S.E.M.) 34 s (n=18), lack of Thr(286)-autophosphorylation of alphaCaMKII however promotes the formation of early transient punctae (peak at 40 s). In contrast, the T286D-mimick of phospho-Thr(286)-alphaCaMKII forms punctae with a delay >10 min, indicating that Thr(286)-autophosphorylation is antagonistic to CaMKII clustering. A two-state model is proposed in which phospho-Thr(286)-alphaCaMKII, formed immediately upon Ca(2+) stimulation, is primarily responsible for target interactions and memory functions of alphaCaMKII. However, a distinct clustering form denoted alphaCaMKII(c), generated upon persistent intracellular free Ca(2+) elevation, is deposited in the punctae which are made of self-interacting CaM/CaMKII complexes. Punctate deposition disables both the interactions and the activity of CaMKII.

Cytochrome b sequence analysis reveals differential molecular evolution in African mole-rats of the chromosomally hyperdiverse genus Fukomys (Bathyergidae, Rodentia) from the Zambezian region.

African mole-rats (Bathyergidae, Rodentia) of the (eu)social genus Fukomys are one of the most speciose mammal genera endemic to Sub-Saharan Africa. Fukomys distributed in the Zambezian phytochorion is characterized by extreme chromosomal variation (2n=40-78). We inferred a molecular phylogeny of Zambezian Fukomys to resolve the interrelationships and the evolutionary history of the known chromosomal races. We sequenced the entire cytochrome b gene (1140bp) for a total of 66 specimens representing 18 karyotypical races from Zambia. An additional 31 sequences were retrieved from GenBank including data on all other chromosomal races. The haplotypes belonging to a small chromosomal race from Salujinga cluster with the Fukomys mechowii (Giant mole-rat) haplotypes. Differential degrees of chromosomal variation are observed among the major mole-rat clades, which is most pertinent when comparing the central Zambezian Fukomys micklemi and the northern Zambezian Fukomys whytei clades. The karyotypically hyper-diverse (12 known chromosomal races) Fukomys micklemi clade shows low levels of cytochrome b sequence divergence. Within the F. whytei clade we find a more conservative pattern of chromosomal diversification (three known chromosomal races) while the levels of sequence divergence are much higher then in the F. micklemi clade. Our results suggest that chromosomal changes may drive phyletic divergence and, eventually, speciation. The observed cladogenetic events during the Plio-Pleistocene within the F. mechowii, F. whytei, F. damarensis and F. micklemi clades appear to coincide with climatically mediated speciation bursts in other savannah dwelling mammals, including hominids. Based on the molecular data presented, combined with morphological and chromosomal data, the taxonomic implication seems to be that Fukomys may contain several (undescribed) cryptic species.

Changes in intake of key nutrients over 17 years during adult life of a British birth cohort.

An investigation was carried out to determine whether there were significant changes in nutrient intake over 17 years of adult life. The Medical Research Council National Survey of Health and Development is a longitudinal study of a nationally representative cohort of singleton births in the UK in 1946. Of this cohort, 1253 survey members provided information on diet recorded in a 5 d diary at age 36 years in 1982, 43 years in 1989 and 53 years in 1999. The outcome measures were mean intakes of energy, macronutrients, minerals and vitamins. There were significant changes in the intake of most nutrients in 1999 compared with previous years. Intakes of fat, Na, Fe and Cu have fallen, but there was a rising trend in the intakes of Ca, P, carotene, thiamin, pyridoxine, folic acid and vitamins C, D and E in both men and women. Additionally, intakes of K, Mg and vitamin K1 have risen in women. There were significant gender differences, women showing a higher percentage rise in the intakes of carotene, riboflavin, folic acid, vitamin C and vitamin E. These changes were related to changes in the consumption of certain key foods, such as the increased consumption of fruit and vegetables and a shift away from whole milk, butter and red meat. Most of these trends are in line with accepted nutritional guidelines. How far these changes are due to consumer choice and real changes in food composition or are due to artefacts inherent in the methodology is discussed.

Comparability of dietary patterns assessed by multiple dietary assessment methods: results from the 1946 British Birth Cohort.

OBJECTIVE: The aim of this study is to evaluate the consistency of dietary patterns assessed through the use of a dietary recall and a 5-day food diary. DESIGN: Participants (n = 2265) of a longitudinal study of health and development completed 48-h dietary recall at interview, followed by a 5-day food diary and with the 24 h immediately preceding the interview analysed separately as a 24-h recall. Mean intakes of foods and nutrients were calculated and dietary patterns were assessed using exploratory factor analysis, using the method of principal components. Paired t-tests and correlation coefficients were used to compare the three dietary assessment methods. RESULTS: Five distinct dietary patterns were identified using the food diary and the 48-h recall but were less consistent on the 24-h recall. Correlations between factor scores on the 48-h recall and the food diary (r = 0.13-0.67) were higher than those between the 24-h recall and food diary (r = -0.01-0.59). The recall methods were effective at ranking subjects according to food and nutrient intakes, with the 48-h recall and food diary showing higher correlations in both males and females. CONCLUSIONS: This study indicates that a 48-h recall effectively characterises dietary patterns in British adults when compared to a food diary and ranks participants appropriately with respect to most nutrients and foods and is superior to a single 24-h recall. These results have implications for longitudinal studies where maximising response rates to repeat dietary assessment tools is essential.

The impact of inter-generational social and regional circumstances on dietary intake patterns of British adults: results from the 1946 British Birth Cohort.

OBJECTIVES: (1) To determine the extent to which an individual's childhood social circumstances and region of residence influence their dietary pattern at age 43 years and (2) to establish the extent to which an individual adopts the dietary pattern of their social and regional circumstances at age 43 years. DESIGN: Longitudinal study of a social class stratified, random sample of all legitimate, singleton births in the week of 3-9 March 1946. SETTINGS: England, Scotland and Wales. SUBJECTS: The 3187 survey members who provided sociodemographic information at age 4 years in 1950 and sociodemographic and dietary data (48-hour dietary recall) at 43 years in 1989. RESULTS: People who remained in the non-manual social class consumed significantly higher amounts of food items correlated with the factor health aware (items include high-fibre breakfast cereals, wholemeal breads, apples and bananas) than those who remained in the manual social class. Those who made the transition from manual social class in childhood to non-manual social class at age 43 years partly adopted the distinctive dietary patterns of the non-manual social classes. Consumption of items in the factors refined (items include whole-fat milk, white bread, sugar and butter) and sandwich (items include tomatoes, lettuce, onions, bacon and ham) did not differ by social class or regional mobility. CONCLUSIONS: This work suggests that although adult dietary patterns are developed as a result of childhood influences, these patterns can be modified as a result of social and regional transitions. Such changes in dietary patterns may influence susceptibility to disease.

Dietary acid-base balance and intake of bone-related nutrients in Cambridge teenagers.

OBJECTIVES: To evaluate the diet of 16-18-y-old boys and girls with particular reference to intakes of nutrients believed to affect bone health and dietary acid-base balance. DESIGN: A 7-day food diary was completed between the months of October and December. SETTING: Cambridge, UK. SUBJECTS: A total of 111 boys and 101 girls aged 16-18 y who were recruited into the Cambridge Bone Studies. MAIN OUTCOME MEASURES: Mean daily intakes of foods and selected nutrients (protein, calcium, phosphorus, magnesium, potassium, vitamins C and K) were calculated. Two estimates of acid-base balance were calculated from the diet using the formulae of Remer (net acid excretion, estimated indirectly; NAE(ind)) and Frassetto (protein/potassium ratio). RESULTS: Mean calcium and phosphorus intakes were above the UK Reference Nutrient intake (RNI). In all, 39% of the boys and 36% of the girls had vitamin K intakes lower than 1 microg/kg body weight/day. Calcium intake was positively correlated with all other nutrients except vitamins C and K. Boys had a significantly higher estimated net acid excretion (NAE(ind)) than girls (P<0.001). Although a strong correlation (r=0.76, P<0.001) was found between the two methods, at higher acid levels a divergence was observed. A significant positive correlation was found between NAE(ind) and the weight consumed per day of milk, cheese, meat and cereal foods and a negative correlation was found with the weight of potatoes and fruit. Diet composition is such that a lower NAE(ind) is accompanied by a lower calcium intake. CONCLUSIONS: The interpretation of the effects of calcium and other nutrients on bone cannot be considered in isolation from the other components of the diet. These results challenge some of the accepted perceptions about what constitutes an optimal diet for the promotion of bone health in adolescents.

Childhood and adult dietary vitamin E intake and cardiovascular risk factors in mid-life in the 1946 British Birth Cohort.

OBJECTIVES: To determine whether dietary vitamin E intake in childhood or mid-life was predictive of adult hypertension and high waist circumference, as two important risk factors for cardiovascular disease. DESIGN: Longitudinal study of a social class stratified random sample of all the legitimate, singleton births in the week of 3-9 March 1946. SETTINGS: England, Scotland and Wales. SUBJECTS: The 2980 survey members who provided information on diet, health and sociodemographic information at two time points; age 4 y in 1950 (24-h dietary recall) and 43 y in 1989 (48-h dietary recall). MAIN OUTCOME MEASURES: Outcomes were adjusted odds ratios (ORs) for hypertension and high waist circumference at age 43 y by thirds of vitamin E intake, relative to the highest intake thirds at both ages. RESULTS: The lowest consumers of vitamin E in both childhood and adulthood were more likely to be hypertensive (OR 1.8, 95% confidence interval (CI): 1.03-3.08) and have high waist circumference (OR 1.6, 95% CI: 1.02-2.43) than those consuming high levels at both ages. A low intake of vitamin E at just one time point was not associated with a statistically significant increased risk of hypertension or high waist circumference. Social class was also an independent and equally strong predictor of these coronary risk factors, indicating that the relation between social class and cardiovascular risks was not mediated solely by the current measures of diet and lifestyle. CONCLUSIONS: Unique data on vitamin E intake from foods in both childhood and adulthood have indicated that relatively low intake of vitamin E at both ages predicted hypertension and high waist circumference at age 43 y.

Intake and sources of phylloquinone (vitamin K1): variation with socio-demographic and lifestyle factors in a national sample of British elderly people.

Intake and sources of phylloquinone (vitamin K1) were examined according to socio-demographic and lifestyle factors in free-living British people aged 65 years and over, from the 1994-5 National Diet and Nutrition Survey. Complete 4-d weighed dietary records were obtained from 1152 participants living in private households. Using newly-available, mainly UK-specific food content data, the weighted geometric mean intake of phylloquinone was estimated at 65 (95 % CI 62, 67) microg/d for all participants, with higher intakes in men than in women (70 v. 61 microg/d respectively, P<0.01). The mean nutrient densities of phylloquinone intake were 9.3 and 10.5 microg/MJ for men and women respectively (P<0.01), after adjusting for age group, region and smoking status. Of all the participants, 59 % had phylloquinone intakes below the current guideline for adequacy of 1 microg/kg body weight per d. Participants aged 85 years and over, formerly in manual occupations, or living in Scotland or in northern England reported lower phylloquinone intakes than their comparative groups. Overall, vegetables contributed 60 % of total phylloquinone intake, with cooked green vegetables providing around 28 % of the total. Dietary supplements contributed less than 0.5 % of phylloquinone intake. Participants living in northern England or in Scotland, in particular, derived less phylloquinone from vegetables than those living in southern England.

Plasma phylloquinone (vitamin K1) concentration and its relationship to intake in a national sample of British elderly people.

Plasma phylloquinone (vitamin K1) concentration was examined according to season, socio-demographic and lifestyle factors and phylloquinone intake in a nationally representative sample of British people aged 65 years and over from the 1994-5 National Diet and Nutrition Survey. Values for both plasma phylloquinone concentration and phylloquinone intake were available from 1076 participants (561 men, 515 women). Eight hundred and thirty-four were living in private households, 242 in residential or nursing homes. Weighted geometric mean plasma phylloquinone concentrations were 0.36 (inner 95% range [corrected] 0.06, 2.01) and 0.24 (inner 94% range [corrected] 0.06, 0.96) nmol/l in free-living and institution samples respectively. Plasma phylloquinone concentrations did not generally differ between men and women, although values in free-living people were significantly lower during autumn and winter (October to March). Plasma phylloquinone concentration was not significantly associated with age. Plasma phylloquinone concentrations were positively correlated with phylloquinone intake in free-living men and women (r 0.18 and 0.30 respectively, both P<0.001). Stepwise multiple regression analysis found that 11 % of the variation in plasma phylloquinone concentration was explained by phylloquinone intake, season and plasma triacylglycerol concentration. After adjustment for age and corresponding nutrient intakes, plasma phylloquinone concentration was significantly associated (each P<0.01) with plasma concentrations of triacylglycerol, cholesterol, retinol and 25-hydroxyvitamin D in free-living women but not men, and with plasma concentrations of carotenes, alpha- and gamma-tocopherols and lutein in free-living men and women. The possibility of concurrent low fat-soluble vitamin status in elderly populations may be a cause for concern.

Leg and trunk length at 43 years in relation to childhood health, diet and family circumstances; evidence from the 1946 national birth cohort.

BACKGROUND: This is a study of the associations of adult leg and trunk length with early life height and weight, diet, socioeconomic circumstances, and health, and parental height, divorce and death. METHOD: The data used were collected in a longitudinal study of the health, development and ageing of a British national birth cohort (N = 2879 in this analysis) studied since birth in 1946. Multiple regression models were used to investigate the relationships. RESULTS: Adult leg and trunk length were each positively associated with parental height, birthweight, and weight at 4 years. Leg length was associated positively with breastfeeding and energy intake at 4 years. Trunk length was associated negatively with serious illness in childhood and possibly also parental divorce, but not with the dietary data. CONCLUSION: Adult leg length is particularly sensitive to environmental factors and diet in early childhood because that is the period of most rapid leg growth. Trunk growth is faster than leg growth after infancy and before puberty, and may be associated with the effects of serious illness and parental separation because of the child's growing sensitivity to stressful circumstances, as well as the result of the biological effects of illness.

Sociodemographic inequalities in the diet of young children in the 1946 British birth cohort.

OBJECTIVE: To evaluate whether there was food and nutrient equality across occupational social classes and geographical region for members of the 1946 British birth cohort at age 4 years. DESIGN: Cross-sectional analysis of selected food groups, energy and nutrients from one-day recall diet records. SETTING: England, Scotland and Wales in 1950. SUBJECTS: Nationally representative sample of 4419 children aged 4 years in 1950 from the MRC National Survey of Health and Development (NSHD) (1946 Birth Cohort). RESULTS: Significant food and nutrient inequalities occurred by region and occupational social class of the father. Disparity in fruit and vegetable consumption primarily led to the nutrient differences, especially with respect to lower vitamin C and carotene intakes in children from Scotland and from a manual social class background. Lower energy intake in Scottish children was attributable to inequality in the consumption of foods providing fat, and also to the retention of the traditional Scottish diet that included porridge and soups. Consumption of some rationed foods -- bacon, orange juice and tea -- was inequitably distributed by father's social class, but others, in particular meat and spreading fats, were consumed more uniformly. In contrast to fruits and vegetables, which showed marked sociodemographic disparities, other non-rationed foods such as bread and potatoes were consumed universally. CONCLUSION: Local cultural norms may have played as strong a part in sociodemographic differences in the diet of children in the early 1950s as did the strict, post-war food rationing that prevailed. In consequence, nutritional equality was not achieved, and the relatively low intake of antioxidant vitamins during early childhood in certain population groups may have compromised health in the long term.

Weight reference charts for British long-term breastfed infants.

AIM: To produce a modification of the British 1990 weight reference reflecting the growth of long-term breastfed infants. METHODS: 120 infants from the Cambridge Infant Growth Study fed breast milk (with no formula) for at least 24 wk, with solids introduced at a mean age of 15 wk, were weighed every 4 wk from birth to 52 wk. Weights were converted to standard deviation scores (SDS) for gender and age post-term based on the revised British 1990 reference, averaged and smoothed across age, and then converted back to weights to provide modified median weight curves by gender. Other centile curves were constructed assuming the same variability and skewness by age as for the British reference. RESULTS: Long-term breastfed infants were slightly heavier than the reference at birth and crossed centiles upwards to reach +0.3 SDS at 2 mo, but subsequently crossed centiles downwards to -0.2 SDS by 12 mo. CONCLUSION: The British 1990 reference, although better than previous growth standards, reflects the growth of long-term breastfed infants only imperfectly, with mean weight falling by 0.5 SDS from 2 to 12 mo. The provision of breastfeeding-specific centiles, either as a transparent overlay or as an alternative chart, should be useful for professionals and parents to monitor the weight of long-term breastfed infants.

Inhibition of HERG potassium channel current by the class 1a antiarrhythmic agent disopyramide.

The Class 1a antiarrhythmic drug disopyramide (DISO) is associated with 'acquired' prolongation of the QT interval of the electrocardiogram (ECG). This potentially proarrhythmic effect is likely to reflect drug actions on ion channels involved in ventricular action potential repolarisation. In this study, we examined the effects of DISO on potassium channels encoded by HERG, as this K channel type has been implicated in both congenital and acquired long-QT syndromes (LQTS). Chinese hamster ovary cells were transiently transfected with HERG cDNA for subsequent whole cell patch clamp recording. HERG tail currents recorded at -40 mV following test pulses to +30 mV were inhibited in a dose-dependent fashion by DISO concentrations within the clinical range (IC50 = 7.23 +/- 0.72 microM; mean +/- SEM). Experiments with 10 microM DISO indicated that the degree of HERG blockade showed some voltage dependence. Further data obtained using an 'envelope of tails' protocol (pulse potential +40 mV) were consistent with a significant role for open-channel blockade at lower drug concentrations. At higher concentrations it is possible that blockade may have involved drug binding to both resting and open channels. Inhibition of the inactivation-deficient mutant HERG-S631A was comparable to that seen for wild-type HERG. Therefore, channel inactivation was not obligatory for DISO to exert its effect. Native delayed rectifier tail currents from rabbit isolated ventricular myocytes were also inhibited by DISO. We conclude (a) that DISO inhibits HERG encoded potassium channels at clinically relevant concentrations and (b) that this action may constitute the molecular basis for acquired LQTS associated with this drug.

Effects of the class III antiarrhythmic agent dofetilide (UK-68,798) on L-type calcium current from rabbit ventricular myocytes.

The methanesulphonanilide agent dofetilide (UK-68,798) exerts Class III antiarrhythmic effects by inhibiting the cardiac rapid delayed rectifier potassium current (I(Kr)) encoded by HERG. The aim of the present study was to determine whether dofetilide also exhibits Class IV (L-type calcium-channel blocking) effects. L-type calcium current (I(Ca,L)) was measured from rabbit isolated ventricular myocytes, using the whole-cell patch-clamp technique under selective recording conditions. Positive control experiments demonstrated inhibition of I(Ca,L) elicited by pulses to + 10 mV by both nifedipine and externally applied Ni2+ ions. Three concentrations of dofetilide were tested: 100 nM, 1 microM and 10 microM. I(Ca,L) magnitude was not significantly reduced by any of the concentrations tested (P > 0.05; n = minimum of seven cells per drug concentration). The inactivation time-course of I(Ca,L) was also unaffected by 10 microM dofetilide. Heterologously expressed HERG current (I(HERG)) recorded from Chinese Hamster Ovary cells was extensively inhibited by 100 nm and 1 microM dofetilide, with inhibition at 1 microM not significantly different from 100% (P > 0.1). It is concluded that dofetilide produced no I(Ca,L) blocking effects at concentrations up to and exceeding that required for maximal I(HERG) inhibition. The findings support the notion that dofetilide is a highly selective Class III antiarrhythmic agent, devoid of Class IV antiarrhythmic activity.

Fat and energy needs of children in developing countries.

The fat requirements of children can be judged according to 4 criteria: 1) the possible obligate needs of fat as a metabolic fuel, 2) the provision of a sufficiently energy-dense diet to meet energy needs, 3) the adequate supply of essential fatty acids, and 4) the supply of sufficient fat to allow adequate absorption of fat-soluble vitamins. In these respects the fat requirements of children in developing countries are probably similar to those of children in affluent nations except for the additional needs imposed by environmental stresses, particularly recurrent infections. In many developing countries, the low energy density of weaning foods appears to be a major contributor to growth faltering and ultimate malnutrition. Evidence from doubly labeled water studies suggests that these diets are adequate when children are healthy but fail to support rapid catch-up growth after diarrhea and other infections. The issues in determining and meeting the fat needs of children in developing countries are illustrated with use of detailed comparative dietary data from a rural community in The Gambia and from Cambridge, United Kingdom. The outstanding feature of the Gambian data is the great importance of breast milk as a source of fat and essential fatty acids up until the end of the second year of life. Weaning foods and adult foods contain low amounts of fat, which causes a sharp transition from adequate fat intakes to probable inadequate fat intakes when children are weaned from the breast. The effects of such low fat intakes, particularly in terms of immune function, require investigation.

Long-term adequacy of exclusive breast-feeding: how scientific research has led to revised opinions.

The present paper reviews the research of the authors and their colleagues over the past 20 years to provide improved nutritional and anthropometric guidelines for the assessment of lactational adequacy and for when the weaning process might be initiated. The nutritional guidelines are based on revised dietary energy requirements. The basic assumption is that since breast milk is a well-balanced food, if energy needs are satisfied so will those for essential nutrients. Energy requirements for young babies have been derived from the application of the doubly-labelled-water technique. This approach indicated that 460 kJ (110 kcal)/kg per d at 1 month and 397 and 355 kJ (95 and 85 kcal)/kg per d at 3 and 6 months respectively would be satisfactory for a nutrient content of high bioavailability. Translated into a breast-milk intake of 850 ml/d the latter would cover the dietary energy needs of the average child growing along the 50th centile until at least 4 months, but the typical child from many developing countries following the 25th centile until 6 months. The importance of revised growth reference values for infancy, equally crucial for assessing lactational adequacy, is also reviewed. In contrast with the shapes of earlier reference patterns, growth trajectories are different when babies are fed in accordance with modern paediatric advice. Mothers and health professionals using the older growth charts to assess the progress of a baby can be misled into assuming that the weaning process needs to be introduced sooner than necessary. Examples of this situation within the context of a developing country are provided.