Effect of Sustainably Sourced Protein Consumption on Nutrient Intake and Gut Health in Older Adults: A Systematic Review.

Diet is integral to the healthy ageing process and certain diets can mitigate prolonged and deleterious inflammation. This review aims to assess the impact of diets high in sustainably sourced proteins on nutrient intake, gut, and age-related health in older adults. A systematic search of the literature was conducted on 5 September 2023 across multiple databases and sources. Studies assessing sustainably sourced protein consumption in community dwelling older adults (≥65 years) were included. Risk of bias (RoB) was assessed using 'RoB 2.0' and 'ROBINS-E'. Narrative synthesis was performed due to heterogeneity of studies. Twelve studies involving 12,166 older adults were included. Nine studies (n = 10,391) assessed habitual dietary intake and had some RoB concerns, whilst three studies (n = 1812), two with low and one with high RoB, conducted plant-based dietary interventions. Increased adherence to sustainably sourced diets was associated with improved gut microbial factors (n = 4640), healthier food group intake (n = 2142), and increased fibre and vegetable protein intake (n = 1078). Sustainably sourced diets positively impacted on gut microbiota and healthier intake of food groups, although effects on inflammatory outcomes and health status were inconclusive. Future research should focus on dietary interventions combining sustainable proteins and fibre to evaluate gut barrier function and consider inflammatory and body composition outcomes in older adults.

Rare variant associations with birth weight identify genes involved in adipose tissue regulation, placental function and insulin-like growth factor signalling.

Investigating the genetic factors influencing human birth weight may lead to biological insights into fetal growth and long-term health. Genome-wide association studies of birth weight have highlighted associated variants in more than 200 regions of the genome, but the causal genes are mostly unknown. Rare genetic variants with robust evidence of association are more likely to point to causal genes, but to date, only a few rare variants are known to influence birth weight. We aimed to identify genes that harbour rare variants that impact birth weight when carried by either the fetus or the mother, by analysing whole exome sequence data in UK Biobank participants. We annotated rare (minor allele frequency <0.1%) protein-truncating or high impact missense variants on whole exome sequence data in up to 234,675 participants with data on their own birth weight (fetal variants), and up to 181,883 mothers who reported the birth weight of their first child (maternal variants). Variants within each gene were collapsed to perform gene burden tests and for each associated gene, we compared the observed fetal and maternal effects. We identified 8 genes with evidence of rare fetal variant effects on birth weight, of which 2 also showed maternal effects. One additional gene showed evidence of maternal effects only. We observed 10/11 directionally concordant associations in an independent sample of up to 45,622 individuals (sign test P=0.01). Of the genes identified, IGF1R and PAPPA2 (fetal and maternal-acting) have known roles in insulin-like growth factor bioavailability and signalling. PPARG, INHBE and ACVR1C (all fetal-acting) have known roles in adipose tissue regulation and rare variants in the latter two also showed associations with favourable adiposity patterns in adults. We highlight the dual role of PPARG in both adipocyte differentiation and placental angiogenesis. NOS3, NRK, and ADAMTS8 (fetal and maternal-acting) have been implicated in both placental function and hypertension. Analysis of rare coding variants has identified regulators of fetal adipose tissue and fetoplacental angiogenesis as determinants of birth weight, as well as further evidence for the role of insulin-like growth factors.

Programming of cardiac metabolism by miR-15b-5p, a miRNA released in cardiac extracellular vesicles following ischemia-reperfusion injury.

OBJECTIVE: We investigated the potential involvement of miRNAs in the developmental programming of cardiovascular diseases (CVD) by maternal obesity. METHODS: Serum miRNAs were measured in individuals from the Helsinki Birth Cohort (with known maternal body mass index), and a mouse model was used to determine causative effects of maternal obesity during pregnancy and ischemia-reperfusion on offspring cardiac miRNA expression and release. RESULTS: miR-15b-5p levels were increased in the sera of males born to mothers with higher BMI and in the hearts of adult mice born to obese dams. In an ex-vivo model of perfused mouse hearts, we demonstrated that cardiac tissue releases miR-15b-5p, and that some of the released miR-15b-5p was contained within small extracellular vesicles (EVs). We also demonstrated that release was higher from hearts exposed to maternal obesity following ischaemia/reperfusion. Over-expression of miR-15b-5p in vitro led to loss of outer mitochondrial membrane stability and to repressed fatty acid oxidation in cardiomyocytes. CONCLUSIONS: These findings suggest that miR-15-b could play a mechanistic role in the dysregulation of cardiac metabolism following exposure to an in utero obesogenic environment and that its release in cardiac EVs following ischaemic damage may be a novel factor contributing to inter-organ communication between the programmed heart and peripheral tissues.

Lifelong dietary protein restriction accelerates skeletal muscle loss and reduces muscle fibre size by impairing proteostasis and mitochondrial homeostasis.

The early life environment significantly affects the development of age-related skeletal muscle disorders. However, the long-term effects of lactational protein restriction on skeletal muscle are still poorly defined. Our study revealed that male mice nursed by dams fed a low-protein diet during lactation exhibited skeletal muscle growth restriction. This was associated with a dysregulation in the expression levels of genes related to the ribosome, mitochondria and skeletal muscle development. We reported that lifelong protein restriction accelerated loss of type-IIa muscle fibres and reduced muscle fibre size by impairing mitochondrial homeostasis and proteostasis at 18 months of age. However, feeding a normal-protein diet following lactational protein restriction prevented accelerated fibre loss and fibre size reduction in later life. These findings provide novel insight into the mechanisms by which lactational protein restriction hinders skeletal muscle growth and includes evidence that lifelong dietary protein restriction accelerated skeletal muscle loss in later life.

Precision gestational diabetes treatment: a systematic review and meta-analyses.

BACKGROUND: Gestational Diabetes Mellitus (GDM) affects approximately 1 in 7 pregnancies globally. It is associated with short- and long-term risks for both mother and baby. Therefore, optimizing treatment to effectively treat the condition has wide-ranging beneficial effects. However, despite the known heterogeneity in GDM, treatment guidelines and approaches are generally standardized. We hypothesized that a precision medicine approach could be a tool for risk-stratification of women to streamline successful GDM management. With the relatively short timeframe available to treat GDM, commencing effective therapy earlier, with more rapid normalization of hyperglycaemia, could have benefits for both mother and fetus. METHODS: We conducted two systematic reviews, to identify precision markers that may predict effective lifestyle and pharmacological interventions. RESULTS: There was a paucity of studies examining precision lifestyle-based interventions for GDM highlighting the pressing need for further research in this area. We found a number of precision markers identified from routine clinical measures that may enable earlier identification of those requiring escalation of pharmacological therapy (to metformin, sulphonylureas or insulin). This included previous history of GDM, Body Mass Index and blood glucose concentrations at diagnosis. CONCLUSIONS: Clinical measurements at diagnosis could potentially be used as precision markers in the treatment of GDM. Whether there are other sensitive markers that could be identified using more complex individual-level data, such as omics, and if these can feasibly be implemented in clinical practice remains unknown. These will be important to consider in future studies.

Gestational diabetes (GDM) is high blood sugar first detected during pregnancy. Normalizing blood sugar levels quickly is important to avoid pregnancy complications. Many women achieve this with lifestyle changes, such as to diet, but some need to inject insulin or take tablets. We did two thorough reviews of existing research to see if we could predict which women need medication. Firstly we looked for ways to identify the characteristics of women who benefit most from changing their lifestyles to treat GDM, but found very limited research on this topic. We secondly searched for characteristics that help identify women who need medication to treat GDM. We found some useful characteristics that are obtained during routine pregnancy care. Further studies are needed to test if additional information could provide even better information about how we could make GDM treatment more tailored for individuals during pregnancy.

The metabolic response of human trophoblasts derived from term placentas to metformin.

AIMS/HYPOTHESIS: Metformin is increasingly used therapeutically during pregnancy worldwide, particularly in the treatment of gestational diabetes, which affects a substantial proportion of pregnant women globally. However, the impact on placental metabolism remains unclear. In view of the association between metformin use in pregnancy and decreased birthweight, it is essential to understand how metformin modulates the bioenergetic and anabolic functions of the placenta. METHODS: A cohort of 55 placentas delivered by elective Caesarean section at term was collected from consenting participants. Trophoblasts were isolated from the placental samples and treated in vitro with clinically relevant doses of metformin (0.01 mmol/l or 0.1 mmol/l) or vehicle. Respiratory function was assayed using high-resolution respirometry to measure oxygen concentration and calculated [Formula: see text]. Glycolytic rate and glycolytic stress assays were performed using Agilent Seahorse XF assays. Fatty acid uptake and oxidation measurements were conducted using radioisotope-labelled assays. Lipidomic analysis was conducted using LC-MS. Gene expression and protein analysis were performed using RT-PCR and western blotting, respectively. RESULTS: Complex I-supported oxidative phosphorylation was lower in metformin-treated trophoblasts (0.01 mmol/l metformin, 61.7% of control, p<0.05; 0.1 mmol/l metformin, 43.1% of control, p<0.001). The proton efflux rate arising from glycolysis under physiological conditions was increased following metformin treatment, up to 23±5% above control conditions following treatment with 0.1 mmol/l metformin (p<0.01). There was a significant increase in triglyceride concentrations in trophoblasts treated with 0.1 mmol/l metformin (p<0.05), particularly those of esters of long-chain polyunsaturated fatty acids. Fatty acid oxidation was reduced by ~50% in trophoblasts treated with 0.1 mmol/l metformin compared with controls (p<0.001), with no difference in uptake between treatment groups. CONCLUSIONS/INTERPRETATION: In primary trophoblasts derived from term placentas metformin treatment caused a reduction in oxidative phosphorylation through partial inactivation of complex I and potentially by other mechanisms. Metformin-treated trophoblasts accumulate lipids, particularly long- and very-long-chain polyunsaturated fatty acids. Our findings raise clinically important questions about the balance of risk of metformin use during pregnancy, particularly in situations where the benefits are not clear-cut and alternative therapies are available.

Large-scale exome sequence analysis identifies sex- and age-specific determinants of obesity.

Obesity contributes substantially to the global burden of disease and has a significant heritable component. Recent large-scale exome sequencing studies identified several genes in which rare, protein-coding variants have large effects on adult body mass index (BMI). Here we extended such work by performing sex-stratified associations in the UK Biobank study (N∼420,000). We identified genes in which rare heterozygous loss-of-function increases adult BMI in women (DIDO1, PTPRG, and SLC12A5) and in men (SLTM), with effect sizes up to ∼8 kg/m2. This is complemented by analyses implicating rare variants in OBSCN and MADD for recalled childhood adiposity. The known functions of these genes, as well as findings of common variant genome-wide pathway enrichment analyses, suggest a role for neuron death, apoptosis, and DNA damage response mechanisms in the susceptibility to obesity across the life-course. These findings highlight the importance of considering sex-specific and life-course effects in the genetic regulation of obesity.

Early life impacts of maternal obesity: a window of opportunity to improve the health of two generations.

The number of pregnancies complicated by obesity is increasing in line with the worldwide obesity crisis; recent estimates suggest that in developed countries more than 50% of pregnancies are in women who are overweight or have obesity. Maternal obesity is associated with an increased risk of many adverse outcomes for both the mother and baby during pregnancy and birth. In addition to these immediate outcomes, maternal obesity before and during pregnancy is associated with an increased risk of offspring cardio-metabolic disease later in life. Studies comparing siblings discordant for in utero exposure to maternal obesity suggest this is not simply due to transmission of 'obesogenic genes' between mother and child or current lifestyle factors, but reflects a direct impact of the obese intrauterine environment on fetal development. This review will describe the long-term consequences of exposure to maternal obesity during development for the cardio-metabolic health of the offspring. It will also discuss the potential molecular mechanisms that underlie the increased risk of metabolic disease in offspring of mothers with obesity, and explore interventions that may be implemented during pregnancy to limit the impact of obesity on offspring long-term health. This article is part of a discussion meeting issue 'Causes of obesity: theories, conjectures and evidence (Part I)'.

Molecular mechanisms underlying adverse effects of dexamethasone and betamethasone in the developing cardiovascular system.

Antenatal glucocorticoids accelerate fetal lung maturation and reduce mortality in preterm babies but can trigger adverse effects on the cardiovascular system. The mechanisms underlying off-target effects of the synthetic glucocorticoids mostly used, Dexamethasone (Dex) and Betamethasone (Beta), are unknown. We investigated effects of Dex and Beta on cardiovascular structure and function, and underlying molecular mechanism using the chicken embryo, an established model system to isolate effects of therapy on the developing heart and vasculature, independent of effects on the mother or placenta. Fertilized eggs were treated with Dex (0.1 mg kg-1 ), Beta (0.1 mg kg-1 ), or water vehicle (Control) on embryonic day 14 (E14, term = 21 days). At E19, biometry, cardiovascular function, stereological, and molecular analyses were determined. Both glucocorticoids promoted growth restriction, with Beta being more severe. Beta compared with Dex induced greater cardiac diastolic dysfunction and also impaired systolic function. While Dex triggered cardiomyocyte hypertrophy, Beta promoted a decrease in cardiomyocyte number. Molecular changes of Dex on the developing heart included oxidative stress, activation of p38, and cleaved caspase 3. In contrast, impaired GR downregulation, activation of p53, p16, and MKK3 coupled with CDK2 transcriptional repression linked the effects of Beta on cardiomyocyte senescence. Beta but not Dex impaired NO-dependent relaxation of peripheral resistance arteries. Beta diminished contractile responses to potassium and phenylephrine, but Dex enhanced peripheral constrictor reactivity to endothelin-1. We conclude that Dex and Beta have direct differential detrimental effects on the developing cardiovascular system.

Solving the conundrum of intra-specific variation in metabolic rate: A multidisciplinary conceptual and methodological toolkit: New technical developments are opening the door to an understanding of why metabolic rate varies among individual animals of a species: New technical developments are opening the door to an understanding of why metabolic rate varies among individual animals of a species.

Researchers from diverse disciplines, including organismal and cellular physiology, sports science, human nutrition, evolution and ecology, have sought to understand the causes and consequences of the surprising variation in metabolic rate found among and within individual animals of the same species. Research in this area has been hampered by differences in approach, terminology and methodology, and the context in which measurements are made. Recent advances provide important opportunities to identify and address the key questions in the field. By bringing together researchers from different areas of biology and biomedicine, we describe and evaluate these developments and the insights they could yield, highlighting the need for more standardisation across disciplines. We conclude with a list of important questions that can now be addressed by developing a common conceptual and methodological toolkit for studies on metabolic variation in animals.

The long-lasting shadow of litter size in rodents: litter size is an underreported variable that strongly determines adult physiology.

BACKGROUND/PURPOSE: Litter size is a biological variable that strongly influences adult physiology in rodents. Despite evidence from previous decades and recent studies highlighting its major impact on metabolism, information about litter size is currently underreported in the scientific literature. Here, we urge that this important biological variable should be explicitly stated in research articles. RESULTS/CONCLUSION: Below, we briefly describe the scientific evidence supporting the impact of litter size on adult physiology and outline a series of recommendations and guidelines to be implemented by investigators, funding agencies, editors in scientific journals, and animal suppliers to fill this important gap.

Towards Precision Medicine in Gestational Diabetes: Pathophysiology and Glycemic Patterns in Pregnant Women With Obesity.

AIMS: Precision medicine has revolutionized our understanding of type 1 diabetes and neonatal diabetes but has yet to improve insight into gestational diabetes mellitus (GDM), the most common obstetric complication and strongly linked to obesity. Here we explored if patterns of glycaemia (fasting, 1 hour, 2 hours) during the antenatal oral glucose tolerance test (OGTT), reflect distinct pathophysiological subtypes of GDM as defined by insulin secretion/sensitivity or lipid profiles. METHODS: 867 pregnant women with obesity (body mass index ≥ 30 kg/m2) from the UPBEAT trial (ISRCTN 89971375) were assessed for GDM at 28 weeks' gestation (75 g oral glucose tolerance test OGTT; World Health Organization criteria). Lipid profiling of the fasting plasma OGTT sample was undertaken using direct infusion mass spectrometry and analyzed by logistic/linear regression, with and without adjustment for confounders. Insulin secretion and sensitivity were characterized by homeostatic model assessment 2b and 2s, respectively. RESULTS: In women who developed GDM (n = 241), patterns of glycaemia were associated with distinct clinical and biochemical characteristics and changes to lipid abundance in the circulation. Severity of glucose derangement, rather than pattern of postload glycaemia, was most strongly related to insulin action and lipid abundance/profile. Unexpectedly, women with isolated postload hyperglycemia had comparable insulin secretion and sensitivity to euglycemic women, potentially indicative of a novel mechanistic pathway. CONCLUSIONS: Patterns of glycemia during the OGTT may contribute to a precision approach to GDM as assessed by differences in insulin resistance/secretion. Further research is indicated to determine if isolated postload hyperglycemia reflects a different mechanistic pathway for targeted management.

Sex-specific effects of maternal metformin intervention during glucose-intolerant obese pregnancy on body composition and metabolic health in aged mouse offspring.

AIMS/HYPOTHESIS: Metformin is increasingly used to treat gestational diabetes (GDM) and pregnancies complicated by pregestational type 2 diabetes or polycystic ovary syndrome but data regarding long-term offspring outcome are lacking in both human studies and animal models. Using a mouse model, this study investigated the effects of maternal metformin intervention during obese glucose-intolerant pregnancy on adiposity, hepatic steatosis and markers of metabolic health of male and female offspring up to the age of 12 months. METHODS: C57BL/6J female mice were weaned onto either a control diet (Con) or, to induce pre-conception obesity, an obesogenic diet (Ob). The respective diets were maintained throughout pregnancy and lactation. These obese dams were then randomised to the untreated group or to receive 300 mg/kg oral metformin hydrochloride treatment (Ob-Met) daily during pregnancy. In male and female offspring, body weights and body composition were measured from 1 month until 12 months of age, when serum and tissues were collected for investigation of adipocyte cellularity (histology), adipose tissue inflammation (histology and quantitative RT-PCR), and hepatic steatosis and fibrosis (histochemistry and modified Folch assay). RESULTS: At 12 months of age, male Ob and Ob-Met offspring showed increased adiposity, adipocyte hypertrophy, elevated expression of proinflammatory genes, hyperleptinaemia and hepatic lipid accumulation compared with Con offspring. Male Ob-Met offspring failed to show hyperplasia between 8 weeks and 12 months, indicative of restricted adipose tissue expansion, resulting in increased immune cell infiltration and ectopic lipid deposition. Female Ob offspring were relatively protected from these phenotypes but Ob-Met female offspring showed increased adiposity, adipose tissue inflammation, hepatic lipid accumulation, hyperleptinaemia and hyperinsulinaemia compared with Con female offspring. CONCLUSIONS/INTERPRETATION: Maternal metformin treatment of obese dams increased offspring metabolic risk factors in a sex- and age-dependent manner. These observations highlight the importance of following up offspring of both sexes beyond early adulthood after interventions during pregnancy. Our findings illustrate the complexity of balancing short-term benefits to mother and child vs any potential long-term metabolic effects on the offspring when prescribing therapeutic agents that cross the placenta.

Postnatal Protein Intake as a Determinant of Skeletal Muscle Structure and Function in Mice-A Pilot Study.

Sarcopenia is characterised by an age-related decrease in the number of muscle fibres and additional weakening of the remaining fibres, resulting in a reduction in muscle mass and function. Many studies associate poor maternal nutrition during gestation and/or lactation with altered skeletal muscle homeostasis in the offspring and the development of sarcopenia. The aim of this study was to determine whether the musculoskeletal physiology in offspring born to mouse dams fed a low-protein diet during pregnancy was altered and whether any physiological changes could be modulated by the nutritional protein content in early postnatal stages. Thy1-YFP female mice were fed ad libitum on either a normal (20%) or a low-protein (5%) diet. Newborn pups were cross-fostered to different lactating dams (maintained on a 20% or 5% diet) to generate three groups analysed at weaning (21 days): Normal-to-Normal (NN), Normal-to-Low (NL) and Low-to-Normal (LN). Further offspring were maintained ad libitum on the same diet as during lactation until 12 weeks of age, creating another three groups (NNN, NLL, LNN). Mice on a low protein diet postnatally (NL, NLL) exhibited a significant reduction in body and muscle weight persisting up to 12 weeks, unlike mice on a low protein diet only prenatally (LN, LNN). Muscle fibre size was reduced in mice from the NL but not LN group, showing recovery at 12 weeks of age. Muscle force was reduced in NLL mice, concomitant with changes in the NMJ site and changes in atrophy-related and myosin genes. In addition, µCT scans of mouse tibiae at 12 weeks of age revealed changes in bone mass and morphology, resulting in a higher bone mass in the NLL group than the control NNN group. Finally, changes in the expression of miR-133 in the muscle of NLL mice suggest a regulatory role for this microRNA in muscle development in response to postnatal diet changes. Overall, this data shows that a low maternal protein diet and early postnatal life low-protein intake in mice can impact skeletal muscle physiology and function in early life while postnatal low protein diet favours bone integrity in adulthood.

Programming by maternal obesity: a pathway to poor cardiometabolic health in the offspring.

There is an ever increasing prevalence of maternal obesity worldwide such that in many populations over half of women enter pregnancy either overweight or obese. This review aims to summarise the impact of maternal obesity on offspring cardiometabolic outcomes. Maternal obesity is associated with increased risk of adverse maternal and pregnancy outcomes. However, beyond this exposure to maternal obesity during development also increases the risk of her offspring developing long-term adverse cardiometabolic outcomes throughout their adult life. Both human studies and those in experimental animal models have shown that maternal obesity can programme increased risk of offspring developing obesity and adipose tissue dysfunction; type 2 diabetes with peripheral insulin resistance and ß-cell dysfunction; CVD with impaired cardiac structure and function and hypertension via impaired vascular and kidney function. As female offspring themselves are therefore likely to enter pregnancy with poor cardiometabolic health this can lead to an inter-generational cycle perpetuating the transmission of poor cardiometabolic health across generations. Maternal exercise interventions have the potential to mitigate some of the adverse effects of maternal obesity on offspring health, although further studies into long-term outcomes and how these translate to a clinical context are still required.

Impact of Metformin Treatment on Human Placental Energy Production and Oxidative Stress.

Metformin is increasingly prescribed in pregnancy, with beneficial maternal effects. However, it is not known how metformin-treatment impacts metabolism and energy production in the developing feto-placental unit. We assessed the human placental response to metformin using both in vivo and in vitro treated samples. trophoblasts were derived from placentas collected from non-laboured Caesarean deliveries at term, then treated in vitro with metformin (0.01 mM, 0.1 mM or vehicle). Metformin-concentrations were measured using liquid-chromatography mass-spectrometry. Oxygen consumption in cultured-trophoblasts was measured using a Seahorse-XF Mito Stress Test. Markers of oxidative-stress were assayed using qRT-PCR. Metformin-transporter mRNA and protein-levels were determined by quantitative RT-PCR and Western-blotting respectively. Metformin concentrations were also measured in sample trios (maternal plasma/fetal plasma/placental tissue) from pregnancies exposed to metformin on clinical-grounds. Maternal and fetal metformin concentrations in vivo were highly correlated over a range of concentrations (R2 = 0.76, p < 0.001; average fetal:maternal ratio 1.5; range 0.8-2.1). Basal respiration in trophoblasts was reduced by metformin treatment (0.01 mM metformin; p < 0.05, 0.1 mM metformin; p < 0.001). Mitochondrial-dependent ATP production and proton leak were reduced after treatment with metformin (p < 0.001). Oxidative stress markers were significantly reduced in primary-trophoblast-cultures following treatment with metformin. There is a close linear relationship between placental, fetal, and maternal metformin concentrations. Primary-trophoblast cultures exposed to clinically-relevant metformin concentrations have reduced mitochondrial-respiration, mitochondrial-dependent ATP-production, and reduced markers of oxidative-stress. Given the crucial role of placental energy-production in supporting fetal growth and well-being during pregnancy, the implications of these findings are concerning for intrauterine fetal growth and longer-term metabolic programming in metformin-exposed pregnancies.

Sex differences in the intergenerational inheritance of metabolic traits.

Strong evidence suggests that early-life exposures to suboptimal environmental factors, including those in utero, influence our long-term metabolic health. This has been termed developmental programming. Mounting evidence suggests that the growth and metabolism of male and female fetuses differ. Therefore, sexual dimorphism in response to pre-conception or early-life exposures could contribute to known sex differences in susceptibility to poor metabolic health in adulthood. However, until recently, many studies, especially those in animal models, focused on a single sex, or, often in the case of studies performed during intrauterine development, did not report the sex of the animal at all. In this review, we (a) summarize the evidence that male and females respond differently to a suboptimal pre-conceptional or in utero environment, (b) explore the potential biological mechanisms that underlie these differences and (c) review the consequences of these differences for long-term metabolic health, including that of subsequent generations.