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Funder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265, Funder: National Institute for Health Research; FundRef: http://dx.doi.org/10.13039/501100000272, Funder: Marker Diagnostics, Funder: Rugby Football Union, Funder: Midland Neuroscience Teaching and Research Fund, Objective: To investigate the role of salivary small non-coding RNAs (sncRNAs) in the diagnosis of sport-related concussion. Methods: Saliva was obtained from male professional players in the top two tiers of England’s elite rugby union competition across two seasons (2017–2019). Samples were collected preseason from 1028 players, and during standardised head injury assessments (HIAs) at three time points (in-game, post-game, and 36–48 hours post-game) from 156 of these. Samples were also collected from controls (102 uninjured players and 66 players sustaining a musculoskeletal injury). Diagnostic sncRNAs were identified with next generation sequencing and validated using quantitative PCR in 702 samples. A predictive logistic regression model was built on 2017–2018 data (training dataset) and prospectively validated the following season (test dataset). Results: The HIA process confirmed concussion in 106 players (HIA+) and excluded this in 50 (HIA−). 32 sncRNAs were significantly differentially expressed across these two groups, with let-7f-5p showing the highest area under the curve (AUC) at 36–48 hours. Additionally, a combined panel of 14 sncRNAs (let-7a-5p, miR-143-3p, miR-103a-3p, miR-34b-3p, RNU6-7, RNU6-45, Snora57, snoU13.120, tRNA18Arg-CCT, U6-168, U6-428, U6-1249, Uco22cjg1,YRNA_255) could differentiate concussed subjects from all other groups, including players who were HIA− and controls, immediately after the game (AUC 0.91, 95% CI 0.81 to 1) and 36–48 hours later (AUC 0.94, 95% CI 0.86 to 1). When prospectively tested, the panel confirmed high predictive accuracy (AUC 0.96, 95% CI 0.92 to 1 post-game and AUC 0.93, 95% CI 0.86 to 1 at 36–48 hours). Conclusions: SCRUM, a large prospective observational study of non-invasive concussion biomarkers, has identified unique signatures of concussion in saliva of male athletes diagnosed with concussion.

Drug delivery by topical application has higher patient acceptance and lower morbidity than intraocular injection, but many ophthalmic treatments are unable to enter the eye or reach the posterior segment after topical application. The first stage towards posterior segment delivery after topical application is ocular surface penetration and existing models are in vivo or use large quantities of tissue. We therefore developed a novel ex vivo model using discs of porcine and human cornea and sclera (5 mm diameter) to assess penetration of a candidate neuroprotective siRNA. siRNA against caspase 2 or control solutions of known penetrance were applied to the corneal epithelial surface and trans-corneal penetration and corneal adsorbance measured at fixed time points. To demonstrate that leakage did not occur, we applied dextran blue, which should not penetrate the intact cornea and did not do so in our model. Fluorescein penetration (0.09%) was less than rhodamine B (6.98%) at 60 min. siCASP2 penetration was 0.01% by 60 min. When the applied siCASP2 was washed off after 2 min, (representing lacrimal drainage) 0.071% penetrated porcine cornea by 60 min and 0.0002% penetrated human cornea and 0.001% penetrated human sclera. Our ex vivo model rapidly and cost-effectively assesses transcorneal penetration of candidate topical therapies, allowing rates of trans-corneal penetration for potential therapies such as siRNA to be evaluated with small quantities of human or animal tissue.

Parthenium hysterophorus L. (Congress grass, family Asteraceae) is found throughout the global agricultural and vacant lands. In the present work, the pharmacognostic, pharmacological and allelopathic traits of various parts (root, stem and leaves) of P. hysterophorus grown in Pakistan were investigated. P. hysterophorus growing/flowering season starts from March–April till August–September. The plant has tap root with numerous rootlets and root hairs. Stem is straight, with light groove, covered with hairs and numerous profusely branches, wherein the stem reaches up to 150 cm in height. The leaves are simple, dull green in color, alternate, leaflets with profusely dissected regions and hairs are present on the lower surface of leaf. Transverse section (TS) of root revealed epidermis, cortex, endodermis, xylem, phloem and medullary rays running from endodermis towards the center. TS of stem was circular in outline with shallow ridges covered with hairs. TS of leaf consisted of epidermis, which is intermitted by simple, glandular, branched and multicellular trichomes. Powder drug microscopy of roots revealed the presence of epidermal cell, phloem elements, fiber, cluster/group of fibers, root hairs, xylem vessels and medullary rays. The stem powder drug microscopy showed the presence of trichomes, fibers, parenchymatous cells, lignified vessel, group/cluster of vessels, secretory gland with epidermal cells, prismatic crystal of Ca oxalate and phloem elements with parenchyma cells. Phytochemical screening of diethyl ether, ethanol and aqueous extracts of root, stem and leaves showed the presence of amino acids, fixed oils, alkaloids, flavonoids, phenols, carbohydrates, proteins and triterpenoids, while steroidal glycosides, gum and mucilage were absent in all plant parts. Chemical composition showed that moisture, lipids, protein contents and total gross energy were higher in plant grown in summer season as compared to winter season. On the other hand, ash, fibers and carbohydrate contents were higher in winter and declined in summer season.

Fibrotic disease is a major cause of mortality worldwide, with fibrosis arising from prolonged inflammation and aberrant extracellular matrix dynamics. Compromised cellular and tissue repair processes following injury, infection, metabolic dysfunction, autoimmune conditions and vascular diseases leave tissues susceptible to unresolved inflammation, fibrogenesis, loss of function and scarring. There has been limited clinical success with therapies for inflammatory and fibrotic diseases such that there remains a large unmet therapeutic need to restore normal tissue homoeostasis without detrimental side effects. We investigated the effects of a newly formulated low molecular weight dextran sulfate (LMW-DS), termed ILB®, to resolve inflammation and activate matrix remodelling in rodent and human disease models. We demonstrated modulation of the expression of multiple pro-inflammatory cytokines and chemokines in vitro together with scar resolution and improved matrix remodelling in vivo. Of particular relevance, we demonstrated that ILB® acts, in part, by downregulating transforming growth factor (TGF)β signalling genes and by altering gene expression relating to extracellular matrix dynamics, leading to tissue remodelling, reduced fibrosis and functional tissue regeneration. These observations indicate the potential of ILB® to alleviate fibrotic diseases.

Ocular chemical injuries (OCIs) commonly cause ocular damage and visual loss and treatment uses topical therapies to facilitate healing and limit complications. However, the impact of chemical injury on corneal barrier function and treatment penetration is unknown. Therefore, the aim of this study was to determine the effect of OCI on drug penetration and absorption. Porcine corneal explants were used to assess histological damage, electrical resistance, and the trans-corneal penetration/corneal adsorption of reference compounds (sodium fluorescein and rhodamine B) and dexamethasone. Corneal explants were injured with either 1 M sulfuric acid, or 1 M sodium hydroxide. Dexamethasone penetration was measured using high-performance liquid chromatography (HPLC) and that of fluorescein and rhodamine using fluorescence. Dexamethasone corneal adsorption was measured using enzyme-linked immunoabsorbant assay (ELISA). Both acid and alkaline injuries reduced trans-corneal electrical resistance. NaOH injury increased hydrophilic fluorescein penetration (NaOH 8.59 ± 1.50E–05 cm.min−1 vs. Hanks' Balanced Salt Solution (HBSS) 1.64 ± 1.01E–06 cm.min−1) with little impact on hydrophobic rhodamine B (1 M NaOH 6.55 ± 2.45E–04 cm.min−1 vs. HBSS 4.60 ± 0.972E–04 cm.min−1) and dexamethasone penetration (1 M NaOH 3.00 ± 0.853E–04 cm.min−1 vs. HBSS 2.69 ± 0.439E–04 cm.min−1). By contrast, H2SO4 decreased trans-corneal penetration of hydrophilic fluorescein (H2SO4 1.16 ± 14.2E–07 cm.min−1) and of hydrophobic dexamethasone (H2SO4 1.88 ± 0.646E–04 cm.min−1) and rhodamine B (H2SO4 4.60 ± 1.42E–05 cm.min−1). Acid and alkaline OCI differentially disrupted the corneal epithelial barrier function. Acid injury reduced penetration of hydrophobic dexamethasone and rhodamine B as well as hydrophilic fluorescein, which may translate clinically into reduced drug penetration after OCI, while alkaline injury increased fluorescein penetration, with minimal effect on dexamethasone and rhodamine B penetration.

Sport-related traumatic brain injury (TBI) elicits a multifaceted inflammatory response leading to brain injury and morbidity. This response could be a predictive tool for the progression of TBI and to stratify the injury of which mild TBI is most prevalent. Therefore, we examined the differential expression of serum inflammatory markers overtime and identified novel markers in repetitively concussed athletes. Neuropsychological assessment by Wechsler Adult Intelligence Scale (WAIS) and Immediate Post Concussion Assessment and Cognitive Test (ImPACT) was performed on rugby players and serum was taken from healthy, concussed and repetitively concussed athletes. Serum was also obtained &lt, 1 week and &gt, 1 week after trauma and analyzed for 92 inflammatory protein markers. Fibroblast growth factor 21 (FGF21) and interleukin-7 (IL-7) differentiated repetitively concussed athletes. Macrophage chemotactic protein-1 (MCP-1), tumor necrosis factor superfamily member 14 (TNFSF14) were significantly reduced &gt, 1 week and chemokine (C-X3-C motif) ligand 1 (CX3CL1) upregulated &lt, 1 week after injury. FGF21 and MCP-1 negatively correlated with symptoms and their severity. We have identified dynamic changes in the inflammatory response overtime and in different classes of concussion correlating with disease progression. This data supports the use of inflammatory biomarkers as predictors of symptom development due to secondary complications of sport-related mTBI.

SummaryThe aim of this article is to explore the leadership qualities of Nelson “Rolihlahla” Mandela in order to find out how his style of leadership can act as a source of inspiration for the Sout...

Corporate social responsibility (CSR) may be defined as the actions of an organisation that are targeted towards achieving a social benefit over and above maximising profits for its shareholders and meeting all its legal obligations (Ghillyer 2009). This definition only scratches the surface of a very complex and often elusive topic that has presented organisations as being the image of unchecked greed. CSR is a phenomenon that is growing in prominence and popularity as organisations find themselves in the limelight due to consumer evolution.

Scholarship on African genocide by African scholars is still in its infancy. Spurred by studies on the Holocaust, African creative writers are slowly but increasingly rendering narratives of African genocide through their fiction. Steadily, new insights are being generated about the banality and evil that surround genocide in an African context and about the harms it causes. However, the original arguments that once made writing about genocide and the pain it inflicts a taboo, appear to continue to haunt the discipline of writing. First, there is the tendency to look down upon the act of writing about genocide experiences, the pretext being that it is in the nature of narrative to distort the density implied in the existential threat to humanity that genocide poses. Second, some literature of atrocity scholars are convinced that human beings who have not experienced genocide first hand are unable to live or even vicariously recreate the agony that genocide survivors experienced and might be able to relate. From this perspective, a genocide canon seems to have been authorised in which it is suggested that only survivors of genocide can produce authentic accounts of the pain that those who died went through. In recent times, critics and creative writers, some of whom are survivors, have challenged the idea of an absolute narrative of genocide by a survivor as the only true bearer of genocide pain and resistance to it. This article explores the panoply of meanings that a genocide survivor, Rupert Bazambanza, gives formal composition to through the genre of cartoon. The article argues that a genocide survivor is an embodiment of a witness and an archive of the experiences of someone who has survived and can tell the stories of the self and also of others who perished.

Background Microbial keratitis is a leading cause of preventable blindness worldwide. Conventional sampling and culture techniques are time-consuming, with over 40% of cases being culture-negative. Nanopore sequencing technology is portable and capable of generating long sequencing reads in real-time. The aim of this study is to evaluate the potential of nanopore sequencing directly from clinical samples for the diagnosis of bacterial microbial keratitis. Methods Using full-length 16S rRNA amplicon sequences from a defined mock microbial community, we evaluated and benchmarked our bioinformatics analysis pipeline for taxonomic assignment on three different 16S rRNA databases (NCBI 16S RefSeq, RDP and SILVA) with clustering at 97%, 99% and 100% similarities. Next, we optimised the sample collection using an ex vivo porcine model of microbial keratitis to compare DNA recovery rates of 12 different collection methods: 21-gauge needle, PTFE membrane (4 mm and 6 mm), Isohelix™ SK-2S, Sugi® Eyespear, Cotton, Rayon, Dryswab™, Hydraflock®, Albumin-coated, Purflock®, Purfoam and Polyester swabs. As a proof-of-concept study, we then used the sampling technique that provided the highest DNA recovery, along with the optimised bioinformatics pipeline, to prospectively collected samples from patients with suspected microbial keratitis. The resulting nanopore sequencing results were then compared to standard microbiology culture methods. Results We found that applying alignment filtering to nanopore sequencing reads and aligning to the NCBI 16S RefSeq database at 100% similarity provided the most accurate bacterial taxa assignment. DNA concentration recovery rates differed significantly between the collection methods (p < 0.001), with the Sugi® Eyespear swab providing the highest mean rank of DNA concentration. Then, applying the optimised collection method and bioinformatics pipeline directly to samples from two patients with suspected microbial keratitis, sequencing results from Patient A were in agreement with culture results, whilst Patient B, with negative culture results and previous antibiotic use, showed agreement between nanopore and Illumina Miseq sequencing results. Conclusion We have optimised collection methods and demonstrated a novel workflow for identification of bacterial microbial keratitis using full-length 16S nanopore sequencing.

The aim of this article is to explore how literary stories are used to teach Muslim children to respect the commands of Allah and the values that undergird Islamic religion. Using biblical allusions drawn from the Koran/Qur’an, stories for young readers such as The Camel and the Evil People (2010) and Stories of the Sahabah for Youth (2011) entertain as well as teach young Muslim readers to appreciate the divine messages of Allah. The lessons that children get from Qur’an stories help them to build character and stick to Muslim principles. They shape personality, test moral uprightness and provide children with a guide to follow the will of Allah. A critical analysis of the stories in the above-mentioned literary texts reveals the multiple layers of themes and perspectives that the texts explore to demonstrate the greatness of Allah and the submissiveness that he expects from his subjects. Through reading Qur’an stories, it is hoped that Muslim children would be empowered to defend Islamic principles, and to distinguish what is right from what is wrong.

Throughout the business world, leaders of the most successful firms have begun to understand the importance of analysing every activity, system and process in their operations to make absolutely sure that they function efficiently and effectively. In the course of this assignment an attempt to understand total quality management will be made, along with an examination of the different schools of thought regarding total quality management and the effect this concept has on production.

The role of regional economic communities in the development of trade in Africa is widely recognised. Currently, intra-African trade stands at 10 per cent. This is in sharp contrast to other developing regions of the world. In Asia and Latin America, the levels of intra-trade are 50 and 26 per cent, respectively. There are a number of reasons accounting for the low level of intra-African trade, including the weak mandate given to regional economic communities to monitor and enforce the commitments assumed by countries under regional trade agreements. The lack of integration has negatively impacted on African countries and affected their ability to attract foreign direct investment commensurate with their development needs. Had African countries been less exposed to external markets, they would have been minimally affected by the global financial crisis. The importance of boosting intra-African trade was highlighted by Africa’s Heads of State and Government when they devoted this year’s summit to this theme. In the run-up to the summit, the African Union Commission released a study that underscored the importance of regional economic communities in the process of economic integration in Africa. Currently, SADC member states are in the process of implementing the SADC Trade Protocol, which would create a fully-fledged free trade area and later a customs union, and at the same time engaged in tripartite negotiations aimed at merging the three (SADC, COMESA and the EAC) regional configurations. They are also engaged in the EPA negotiations with the European Union, which would create a free trade area and also the Doha negotiations under the auspices of the WTO. The main objective of this article is to estimate SADC countries’ bilateral trade potential, which may result in the improvements in trade facilitation.

SummaryThis article engages North African Women's writing their unique identities in the epistolary form as depicted in the anthology, Women Writing Africa: The Northern Region. The epistolary or letter form is a unique genre. On one hand, the form of a letter is personal, and lays claim to private experiences whose content is the confidential lives of those who write them. And yet, on the other hand, the contents of an epistolary form can be thrust into the public domain where the lives on which it narrates is read by many people for the interest it can generate when the form assumes the figure of a metaphoric allegory. In the anthology, there are six letters written by women, touching on themes of slavery, sisterhood, marginality and the quest for political freedom in a patriarchal-dominated North African community. It is curious to observe that North African women who represented their experiences in the form of the letters did so against the repression of this form from the predominance of realism, so...

Many African youths can learn moral values through the oral tradition of folktales as these narratives are used as vehicles to communicate such values. In the past, the oral tradition was the main method of passing on beliefs from generation to generation by word of mouth. Technological and other developments, especially the invention of the book and the increase of literacy, have had an important effect on African oral traditions as previously unwritten folktales could be accorded permanent existence in the form of books. This has afforded African youths in different geographical locations the opportunity to access in their own time the oral folktale and the universal values that it communicates in the written form. Oral forms, such as folktales, are created and developed in specific contexts by individuals, and these can now be experienced by readers in different locations. The aim of this article is to highlight what African youths in different parts of Africa can gain from being exposed to folktales in their written form. For this purpose, four folktale stories by Greaves (1988) contained in the anthology, When Hippo Was Hairy: And Other Tales from Africa, were analysed. The selected stories are representative of certain thematic threads intended to impart certain moral values.

Purpose To determine if vitreous levels of the pro-fibrotic cytokine transforming growth factor beta2 (TGF-β2) and its opposing regulator decorin predict subsequent proliferative vitreoretinopathy (PVR) development in patients with rhegmatogenous retinal detachment (RRD). Methods We examined the effect of TGF-β2 and decorin on epithelial-mesenchymal transition (EMT) and collagen expression in vitro using ARPE-19 cells, and we analyzed extracellular matrix marker expression in PVR membrane and internal limiting membrane patient samples. We performed a prospective noninterventional cohort study, recruiting 125 patients undergoing vitrectomy for RRD and macular hole surgery, measured vitreous levels of TGF-β2 and decorin by ELISA, and followed them up for 6 months. Patients who did not develop PVR were compared to those who did, in order to determine whether vitreous TGF-β2 and decorin levels predicted PVR development. Results In vitro, TGF-β2 induced EMT and collagen production. Decorin strongly inhibited EMT and collagen production at high levels. PVR membranes expressed high levels of fibrosis-associated proteins, consistent with EMT. Vitreous TGF-β2 levels were unchanged between patients with macular holes and RRD who did or did not subsequently develop PVR. Average decorin levels were higher in the vitreous of RRD patients who subsequently developed PVR compared to those who did not, but at the measured vitreous concentrations (1-2 μg/mL), decorin did not demonstrate an in vitro inhibitory effect on EMT. Conclusions In vitro, high concentrations of decorin inhibited EMT and fibrosis. At the levels seen in human vitreous, decorin did not prevent fibrosis or EMT in vitro, and higher initial vitreous decorin levels were associated with the development of postoperative PVR after vitrectomy to treat RRD, but did not reliably predict the outcome.

Recent in silico analysis has revealed the presence of a group of proteins in pro and lower eukaryotes, but not in Man, that show extensive amino acid sequence similarity to known O6-alkylguanine-DNA alkyltransferases, but where the cysteine at the putative active site is replaced by another residue, usually tryptophan. Here we review recent work on these proteins, which we designate as alkyltransferase-like (ATL) proteins, and consider their mechanism of action and role in protecting the host organisms against the biological effects of O6-alkylating agents, and their evolution. ATL proteins from Escherichia coli (eAtl, transcribed from the ybaz open reading frame) and Schizosaccharomyces pombe (Atl1) are able to bind to a range of O6-alkylguanine residues in DNA and to reversibly inhibit the action of the human alkyltransferase (MGMT) upon these substrates. Isolated proteins were not able to remove the methyl group in O6-methylguanine-containing DNA or oligonucleotides, neither did they display glycosylase or endonuclease activity. S. pombe does not contain a functional alkyltransferase and atl1 inactivation sensitises this organism to a variety of alkylating agents, suggesting that Atl1 acts by binding to O6-alkylguanine lesions and signalling them for processing by other DNA repair pathways. Currently we cannot exclude the possibility that ATL proteins arose through independent mutation of the alkyltransferase gene in different organisms. However, analyses of the proteins from E. coli and S. pombe, are consistent with a common function.

The prevalence of obesity in adults and children has increased globally at an alarming rate. Mounting evidence from both epidemiological studies and animal models indicates that adult obesity and associated metabolic disorders can be programmed by intrauterine and early postnatal environment- a phenomenon known as "fetal programming of adult disease." Data from nutritional intervention studies in animals including maternal under- and over-nutrition support the developmental origins of obesity and metabolic syndrome. The hypothalamic neuronal circuits located in the arcuate nucleus controlling appetite and energy expenditure are set early in life and are perturbed by maternal nutritional insults. In this review, we focus on the effects of maternal nutrition in programming permanent changes in these hypothalamic circuits, with experimental evidence from animal models of maternal under- and over-nutrition. We discuss the epigenetic modifications which regulate hypothalamic gene expression as potential molecular mechanisms linking maternal diet during pregnancy to the offspring's risk of obesity at a later age. Understanding these mechanisms in key metabolic genes may provide insights into the development of preventative intervention strategies.

The placenta occupies a critical place in fetal programming by modulating fetal responses to alterations in the maternal environment, by regulating transfer of nutrients between mother and fetus and by responding to changes in environmental stimuli that affect maternal and/or fetal physiology. Placental size and function are exquisitely sensitive to alterations in maternal nutrition, oxygen tension and exposure to glucocorticoids. These factors affect the activity of proteins such as the System A transporter that shuttles neutral amino acids to the fetus, the activity of cells such as those producing placental lactogen, a key metabolic hormone of pregnancy, and the generation of peptides such as the urocortins, members of the corticotrophin releasing hormone family with pro-and anti-inflammatory activities, and placental 11βhydroxysteroid dehydrogenase-2, which regulates placental metabolism of maternal cortisol. Maternal administration of exogenous synthetic glucocorticoid bypasses this metabolic barrier, programming reductions in fetal body weight and composition, and changing development of the fetal neurologic, pancreatic and hypothalamic-pituitary-adrenal (HPA) axes, leading to altered stress and inflammatory responses, cardiovascular activity and predisposition to insulin resistance in later life. Interestingly, many of these placental functions appear to be regulated in a manner that is dependent upon the sex of the fetus.

Epidemiological data indicate that an adverse maternal environment during pregnancy predisposes offspring to metabolic syndrome with increased obesity, and type 2 diabetes. The mechanisms are still unclear although epigenetic modifications are implicated and the hypothalamus is a likely target. We hypothesized that maternal undernutrition (UN) around conception in sheep would lead to epigenetic changes in hypothalamic neurons regulating energy balance in the offspring, up to 5 years after the maternal insult. We found striking evidence of decreased glucocorticoid receptor (GR) promoter methylation, decreased histone lysine 27 trimethylation, and increased histone H3 lysine 9 acetylation in hypothalami from male and female adult offspring of UN mothers. These findings are entirely compatible with the increased GR mRNA and protein observed in the hypothalami. The increased GR predicted the decreased hypothalamic proopiomelanocortin expression and increased obesity that we observed in the 5-year-old adult males. The epigenetic and expression changes in GR were specific to the hypothalamus. Hippocampal GR mRNA and protein were decreased in UN offspring, whereas pituitary GR was altered in a sex-specific manner. In peripheral polymorphonuclear leukocytes there were no changes in GR methylation or protein, indicating that this epigenetic analysis did not predict changes in the brain. Overall, these results suggest that moderate changes in maternal nutrition, around the time of conception, signal life-long and tissue-specific epigenetic alterations in a key gene regulating energy balance in the hypothalamus.

Undernutrition during pregnancy is implicated in the programming of offspring for the development of obesity and diabetes. We hypothesized that maternal programming causes epigenetic changes in fetal hypothalamic pathways regulating metabolism. This study used sheep to examine the effect of moderate maternal undernutrition (60 d before to 30 d after mating) and twinning to investigate changes in the key metabolic regulators proopiomelanocortin (POMC) and the glucocorticoid receptor (GR) in fetal hypothalami. Methylation of the fetal hypothalamic POMC promoter was reduced in underfed singleton, fed twin, and underfed twin groups (60, 73, and 63% decrease, respectively). This was associated with reduced DNA methyltransferase activity and altered histone methylation and acetylation. Methylation of the hypothalamic GR promoter was decreased in both twin groups and in maternally underfed singleton fetuses (52, 65, and 55% decrease, respectively). This correlated with changes in histone methylation and acetylation and increased GR mRNA expression in the maternally underfed singleton group. Alterations in GR were hypothalamic specific, with no changes in hippocampi. Unaltered levels of OCT4 promoter methylation indicated gene-specific effects. In conclusion, twinning and periconceptional undernutrition are associated with epigenetic changes in fetal hypothalamic POMC and GR genes, potentially resulting in altered energy balance regulation in the offspring.—Begum, G., Stevens, A., Smith, E. B., Connor, K., Challis, J. R. G., Bloomfield, F., White, A. Epigenetic changes in fetal hypothalamic energy regulating pathways are associated with maternal undernutrition and twinning.

OBJECTIVE AMP-activated protein kinase (AMPK) signaling acts as a sensor of nutrients and hormones in the hypothalamus, thereby regulating whole-body energy homeostasis. Deletion of Ampkα2 in pro-opiomelanocortin (POMC) neurons causes obesity and defective neuronal glucose sensing. LKB1, the Peutz-Jeghers syndrome gene product, and Ca2+-calmodulin–dependent protein kinase kinase β (CaMKKβ) are key upstream activators of AMPK. This study aimed to determine their role in POMC neurons upon energy and glucose homeostasis regulation. RESEARCH DESIGN AND METHODS Mice lacking either Camkkβ or Lkb1 in POMC neurons were generated, and physiological, electrophysiological, and molecular biology studies were performed. RESULTS Deletion of Camkkβ in POMC neurons does not alter energy homeostasis or glucose metabolism. In contrast, female mice lacking Lkb1 in POMC neurons (PomcLkb1KO) display glucose intolerance, insulin resistance, impaired suppression of hepatic glucose production, and altered expression of hepatic metabolic genes. The underlying cellular defect in PomcLkb1KO mice involves a reduction in melanocortin tone caused by decreased α-melanocyte–stimulating hormone secretion. However, Lkb1-deficient POMC neurons showed normal glucose sensing, and body weight was unchanged in PomcLkb1KO mice. CONCLUSIONS Our findings demonstrate that LKB1 in hypothalamic POMC neurons plays a key role in the central regulation of peripheral glucose metabolism but not body-weight control. This phenotype contrasts with that seen in mice lacking AMPK in POMC neurons with defects in body-weight regulation but not glucose homeostasis, which suggests that LKB1 plays additional functions distinct from activating AMPK in POMC neurons.

Maternal undernutrition is associated with programming of obesity in offspring. While previous evidence has linked programming to the hypothalamic, pituitary, and adrenal (HPA) axis it could also affect the hypothalamic neuropeptides which regulate food intake and energy balance. Alpha melanocyte stimulating hormone (αMSH), a key regulator of these neuronal pathways, is derived from pro-opiomelanocortin (POMC) which is therefore a prime target for the programming of obesity. Several models of maternal undernutrition have identified changes in POMC in hypothalami from foetuses or offspring at various ages. These models have also shown that the offspring go on to develop obesity and/or glucose intolerance. It is our hypothesis that programming leads to epigenetic changes in hypothalamic neuropeptide genes. Therefore when there is subsequent increased food availability, the epigenetic changes could cause dysfunctional transcriptional regulation of energy balance. We present evidence of epigenetic changes in the POMC gene promoter in foetal hypothalami after peri-conceptional undernutrition. In this model there are also epigenetic changes in the hypothalamic glucocorticoid receptor with consequent up-regulation of the receptor which could lead to alterations in the regulation of POMC and neuropeptide Y (NPY) in the hypothalamus. Thus maternal undernutrition could cause epigenetic changes in the POMC and glucocorticoid receptor genes, in the foetal hypothalamus, which may predispose the offspring to altered regulation of food intake, energy expenditure and glucose homeostasis, later in life.