The tumor suppressor p53 is a complex cell signaling hub encompassing multiple transcription programs and governs a vast repertoire of biological responses. However, despite several decades of research, how p53 selects one program over another is still elusive. Recent attempts have used meta-analyses of p53 ChIP-seq data to determine the core p53 transcriptional program, conserved across different models and stimuli. This review highlights the complexity of the multiple layers of p53 regulation and the context specificity of p53 target genes. More specifically, we discuss the controversy over the mechanisms of p53-dependent transcriptional repression and its potential role in the flexibility of p53 response.p53 is a major tumor suppressor that integrates diverse types of signaling in mammalian cells. In response to a broad range of intra- or extra-cellular stimuli, p53 controls the expression of multiple target genes and elicits a vast repertoire of biological responses. The exact code by which p53 integrates the various stresses and translates them into an appropriate transcriptional response is still obscure. p53 is tightly regulated at multiple levels, leading to a wide diversity in p53 complexes on its target promoters and providing adaptability to its transcriptional program. As p53-targeted therapies are making their way into clinics, we need to understand how to direct p53 towards the desired outcome  selectively in cancer cells without affecting normal tissues or the immune system. While the core p53 transcriptional program has been proposed, the mechanisms conferring a cell type- and stimuli-dependent transcriptional outcome by p53 require further investigations. The mechanism by which p53 localizes to repressed promoters and manages its co-repressor interactions is controversial and remains an important gap in our understanding of the p53 cistrome. We hope that our review of the recent literature will help to stimulate the appreciation and investigation of largely unexplored p53-mediated repression. The transcription factor p53 is the most frequently mutated gene in cancer and arguably the most critical barrier against tumorigenesis. Because of its crucial importance in most types of cancer, it has become one of the most studied genes . The higWhile transcription-independent functions of p53 that participate in its pro-apoptotic properties have been described , the priRecently, several studies tried to simplify the complexity of the p53 response toward its core target genes by performing a meta-analysis of p53 cistrome and transcriptome in multiple cell lines upon different stimuli. The authors of these studies suggest that the direct p53 core genes involve a certain number of activated target genes, while p53-dependent repression is only indirect, occurring via induction of p21 followed by activation of the downstream DREAM repressive complex  ,7. In thp53 induces a remarkable variety of biological responses, ranging from transient or permanent growth arrest (senescence), induction of pro-oxidant response and apoptotic cell death , modulatReactivation of the p53 killer function  in established cancers is the ultimate goal of p53-based therapies. However, the induction of p53-mediated apoptosis in normal tissues is the cause of toxicity upon chemotherapy and the pathological loss of cells in neurodegenerative diseases, ischemia and stroke. Induction of senescence by p53 might also contribute to aging . On the p53 is a transcription factor that binds to its response elements (RE) in target genes and facilitates or prevents the formation of active transcription complexes, thus activating or repressing gene expression. p53 transcriptional activity is tightly regulated at multiple levels .p53 is heavily regulated by more than 300 different posttranslational modifications (PTMs), such as phosphorylation, acetylation, ubiquitination, neddylation, SUMOylation and methylation (reviewed in ). PTMs hOn top of PTM complexity, the TP53 gene can give rise to twelve different protein isoforms by a combination of alternative splicing, usage of IRES and alternative promoter . All p53Another level of regulation is the persistence of p53 on the promoter. Several studies have highlighted the importance of p53 dynamics for its transcriptional response ,37,38. AOverall, a network of multiple factors affects p53 transcriptional response, but our understanding of p53 life or death decisions constitutes only the tip of the iceberg. More systematic studies are required to address these urgent questions of p53 biology and provide new ideas for combination therapies to direct p53 response to the desired outcome.ca 100 p53 target genes have been recently defined by comparing multiple complementary datasets. These are induced irrespective of PTMs and biological outcomes (growth arrest/senescence) upon nutlin treatment in three cancer cell lines of different origins [Ccng1, Eda2r, Gtse1, Mdm2, Polk, Psrc1, Zfp365), while the canonical p53 target gene p21 was not among them. This extreme context specificity of the p53 response illustrates how the definition of the core p53 program is challenging and may be biologically irrelevant in a broader picture.Our analysis of genome-wide chromatin occupancy by p53 using ChIP-seq revealed the “p53 default program” , irrespective of the activating compound  and regardless of distinct transcriptional and biological responses triggered by these compounds . The cor origins . The autOverall, considerable evidence supports the notion that the diverse p53-mediated transcriptional programs are governed by the epigenetic state of the chromatin and thus the availability of p53 RE, its PTMs and the repertoire of co-regulators, which greatly vary among cell types and upon different stresses ,42.Thus, genes involved in ‘non-core functions’, including p53-repressed genes, most probably determine the extraordinary flexibility of the context-specific p53 response and drive its tumor suppressor properties in different tissues.p21). The direct repression by p53 involves binding to the gene and recruitment of co-repressors/HDACs or competition with/sequestration of transcriptional activators (reviewed in [The molecular mechanism of p53-mediated gene repression is a subject of active debate . p53 caniewed in ). Despitiewed in ,44). Antiewed in ,46.Another interesting possibility is that p53-dependent repression could be mediated by epigenetic silencing. Indeed, p53 has been shown to regulate the transcription of the DNA methyltransferases DNMT1, DNMT3a and DNMT3b, and the TET enzymes Tet1 and Tet2; hence, p53 can regulate DNA methylation homeostasis . FurtherDrosophila and zebrafish models, demonstrating its importance during evolution [A gene is regarded as a direct p53 target if it matches at least three of the four criteria: (1) p53-dependent differential expression; (2) the presence of p53 response element (RE) in the promoter; (3) p53 binding to its RE; (4) p53-dependent regulation of the Luc reporter containing p53 RE from the gene. Based on these strict criteria, at least two dozen p53-repressed genes have been defined as direct targets . A few evolution .However, in recent years, several studies have suggested that p53 represses genes solely indirectly through the induction of CDK inhibitor p21, which prevents phosphorylation of Rb and Rb-like proteins, thus transmitting the signal from p53 to a repressor DREAM complex ,59 Figuc. Among The major limitation of meta-analysis is that it considers only those peaks and genes that are reproduced in all data sets, disregarding lineage- and stress type-specific gene regulation by p53—since these, by definition, are not common for all datasets, and therefore appear to have lower p53 signal in meta-analysis. Thus, by no means does this meta-analysis of different datasets identify the whole p53 cistrome and transcriptome. These studies have identified a core, or default, p53 chromatin occupancy pattern and transcriptional program, which are triggered in all types of cells irrespective of cellular chromatin state, the nature of activating signal, p53 PTMs and diverse biological outcome. As mentioned above, all these factors create the diversification of context-dependent p53 transcriptional regulation, which needs to be investigated to enable us to manipulate p53 for a long cancer-free life.We have previously analyzed p53 occupancy at repressed genes and noted that p53 peaks are usually smaller on repressed genes in comparison to activated ones . We suggA comprehensive meta-analysis of ChIP-seq data from different experiments remains a challenge; as such, most computational methods consider only the top few hundred highest peaks with the highest affinity RE, whereas low peaks/weaker RE are completely ignored. Nevertheless, several studies have highlighted the role of low-affinity REs for robustness, specificity and precision of gene regulation (reviewed in ).Previous studies (reviewed in ), as welNotably, analysis of the p53 REs sequence in repressed genes showed more flexible dinucleotide combinations in the core CWWG of p53 RE compared to p53-activated genes . ImportaInterestingly, the lower affinity binding sites might require a higher level transcription factor to achieve a productive binding, as shown for another transcription factor—c-Myc, which can activate and repress genes. c-Myc occupancy at repressed genes is lower than that at activated genes and requires higher concentrations of c-Myc for transcriptional repression . This imRecently, a thorough meta-analysis of 46 p53 ChIP-seq datasets has found that only 14% of genes bound and repressed by p53 are DREAM targets . By consIn summary, to identify genes involved in the diversification of the p53 transcriptional response in a signal- and lineage-dependent manner, we need to consider lower affinity/lower occupancy p53 binding sites, including those in p53-repressed genes. More systematic studies are required to elucidate the difference between the p53 RE in activated versus repressed genes. It is possible, for example, that the presence of another transcription factor at the promoter due to a composite binding site  can switch p53 activity towards transcriptional repression. Interestingly, our bioinformatics search for transcription factors with composite binding sites in p53-repressed promoters has identified several of those, including E2F family members (unpublished data). We speculate that activated p53 and E2F bound to hypophosphorylated Rb might cooperate to repress genes. Further studies will hopefully help clarify these issues and identify cellular cofactors and small molecules that can switch p53 activity from gene activation to repression in a controlled fashion.


Numerous studies indicate that zinc and the new zinc-related adipokine, zinc-α2-glycoprotein (ZAG), are involved in lipid metabolism. Excess body fat lowers blood concentrations of Zn and ZAG, leading not only to the development of obesity but also to other components of the metabolic syndrome. Zinc homeostasis disorders in the body negatively affect the lipid profile and cytokine secretion. Zinc appears to be a very important ZAG homeostasis regulator. The physiological effects of ZAG are related to lipid metabolism, but studies show that ZAG also affects glucose metabolism and is linked to insulin resistance. ZAG has a zinc binding site in its structure, which may indicate that ZAG mediates the effect of zinc on lipid metabolism. The review aimed to verify the available studies on the effects of zinc and ZAG on lipid metabolism. A literature review within the scope of this research area was conducted using articles available in PubMed (including MEDLINE), Web of Science and Cochrane Library databases. An analysis of available studies has shown that zinc improves hepatic lipid metabolism and has an impact on the lipid profile. Numerous studies have found that zinc supplementation in overweight individuals significantly reduced blood levels of total cholesterol, LDL (Low-density lipoprotein)cholesterol and triglycerides, potentially reducing cardiovascular morbidity and mortality. Some results also indicate that it increases HDL-C (High-density lipoprotein) cholesterol levels. ZAG has been shown to play a significant role in reducing obesity and improving insulin sensitivity, both in experimental animal model studies and in human studies. Furthermore, ZAG at physiologically relevant concentrations increases the release of adiponectin from human adipocytes. In addition, ZAG has been shown to inhibit in vitro leptin production. Further studies are needed to provide more data on the role of zinc and zinc-α2-glycoprotein. Zinc, as a trace element, plays an important role in human life. It is involved in the growth and development of the body and, as a catalyst for enzymes, participates in the metabolism of lipids, proteins and carbohydrates. It regulates the expression and activation of many molecules and builds over three hundred metalloenzymes. This metal is involved in the regulation of inflammations by affecting pro-inflammatory cytokines and plays a role in reducing oxidative stress. Zinc deficiency impairs growth, reproduction, metabolism and immunity in both humans and animals and may predispose those affected to the development of obesity and other metabolic diseases ,2,3,4. AThis paper constitutes a literature review of 196 articles available in PubMed (including MEDLINE), Web of Science and Cochrane Library databases. The inclusion criterion for papers was information on the role of zinc and/or ZAG in lipid metabolism. Keywords used to search for papers were “zinc”, “zag”, “zinc-α2-glycoprotein”, “lipids” “lipid metabolism”, “adipose tissue” and “obesity” . ExperimZinc (Zn) is currently one of the most important micronutrients in the human body. It is also an essential part of life processes, bone development and body growth.Zinc plays a major role in the metabolism of carbohydrates, fats and proteins. It is a component of more than three hundred metalloenzymes and exhibits antioxidant activity, thus participating in the reduction of oxidative stress. Zinc is also involved in the synthesis, storage and transport of insulin ,6,8. TheZinc deficiency in the body means that the energy production process is disrupted. This is due to the abnormal behaviour of metalloenzymes that include zinc, such as carbonic anhydrase. It is involved in energy production reactions in the body. When this process is disrupted, fat tissue is formed instead of energy, which promotes the development of overweight and obesity. Studies also show that at low blood levels of this element, lipid management is disrupted leading to an increase in total cholesterol, triglycerides and LDL cholesterol ,10.Zinc is a very important regulator of ZAG homeostasis, which plays a role in lipid metabolism and glucose homeostasis . In addiZAG contains trace elements such as zinc. ZAG has been shown to have 2 strong and 15 weak zinc binding sites, and the attachment of zinc at these sites enables ZAG to bind to fatty acids and β-adrenergic receptors. Correct blood zinc concentrations are also essential to maintain adequate ZAG activity, as Zn facilitates the binding of adipokine to substrates. In vitro research has shown zinc binding to play a key role as it induces oligomerisation of the zinc-α2 glycoprotein, allowing ZAG to bind to fatty acids ,13,14,15Zinc-α2 glycoprotein is a polypeptide with a molecular weight of 43 kDa. It was first isolated from human plasma in 1961 . ZAG is The structure of ZAG is similar to major histocompatibility complex (MHC) class 1 proteins. Similar to MHC class I molecules, ZAG has an open groove between its A1 and A2 helix domains. The ZAG groove contains polyethylene glycol (PEG). Calorimetry and fluorescence spectroscopy have shown that the ZAG groove can bind hydrophobic ligands, especially polyunsaturated fatty acids ,22,23.ZAG has been classified as a novel adipokine because it is a lipid mobilising factor . StudiesZAG also affects glucose metabolism and is linked to insulin resistance (IR) . IntraveZAG is also used as a tumour marker as its overexpression is found in several types of malignant cancers ,29,30. OThe ZAG impact mechanism on lipid metabolism has not yet been clearly defined, but the overwhelming majority of studies indicate that ZAG may affect this process in multiple ways .ZAG increases lipolysis in white adipose tissue (WAT) by acting through the classical cyclic AMP pathway . It stimPhysiologically, ZAG plasma levels range from a few mg/dL in a foetus, 8–12 mg/dL in young people, and 18–30 mg/dL in healthy adults and older men . ZAG is The expression of ZAG in adipose tissue changes depending on various factors. Increased expression is influenced by PPARγ, glucocorticoids, some β3-adrenergic receptor agonists, thyroid hormones and growth hormone (GH), among others. On the other hand, chronic inflammation and increased serum leptin levels may reduce ZAG secretion in the adipose tissue ,27,39.Animal studies by Tinkov et al.  showed tLi et al.  in theirp < 0.01) and alanine transaminase (p < 0.05). Oral zinc supplementation reversed the effects of the HFD diet. Zinc reduced triglyceride levels and raised HDL-C cholesterol levels, but did not affect changes in total cholesterol and LDL-C cholesterol. In addition, it decreased the expression of high-sensitivity C-reactive protein (hs-CRP) and interleukin-6. The results of these studies suggest that zinc may protect the liver and the cardiovascular function subject to an unhealthy, high-fat diet.A study by Xu et al.  suggestsStudies show that people with excessive body fat have reduced levels of zinc and ZAG in their blood ,54,55,56Studies by Cayir et al.  found thAs shown by Voruganti et al. , even sh2, p = 0.030 and 90.4 ± 15.4 vs. 88.7 ± 15 kg, p = 0.020, respectively) and highly significant reductions in blood triglyceride levels . On the other hand, in a study by Kelishadi et al. [Findings indicate that impaired Zn homeostasis in obese individuals also affects circulating lipid concentrations in the blood . Numeroui et al. , supplemZinc is also involved in regulating the expression of pro-inflammatory cytokines produced by adipocytes . Zinc adZinc-α2 glycoprotein plays a huge role in the regulation of adipose tissue mass. ZAG acts multidirectionally, playing a role in stimulating lipolysis, inhibiting lipid accumulation in adipose tissue, regulating serum lipid values and influencing the secretion of other adipokines .The results of ongoing studies suggest that ZAG expression in adipocytes is inversely related to fat mass. In a study by Bing et al. , it was Adipocytes and liver cells are major AZGP1 expression sites in rodents . AdminisResearch by Gong et al.  clearly Fan et al. reached similar conclusions . They foFurthermore, evidence from Gao et al.  indicateRussell et al. also observed ZAG impact on adipose tissue browning . Ob/ob mIn contrast, Rolli et al.  showed tStudies by Mracek et al.  and CepeWhite adipose tissue is found in excess in obese and overweight people. Studies indicate that ZAG has the potential to induce WAT browning in 3T3-L1 adipocytes. Xiao et al.  observedIt turns out that ZAG is also found in cord blood and influences the development of body fat as early as infancy. In the study by Näf et al.  ZAG in cp < 0.001) and that they correlated positively with age, waist circumference and BMI, fasting insulin, indices of insulin resistance, adipocyte and fatty acid binding protein (A-FABP), serum TG, and CRP. That research provided the first evidence that the excessive elevation of ZAG in individuals with metabolic disorders and obesity may be a compensatory regulation of the body to the oxidative stress present. Elevated ZAG levels may also indicate ZAG resistance, similar in mechanism to hyperinsulinaemia and hyperleptinaemia caused by the body’s resistance to insulin or leptin. These studies suggest that the serum ZAG concentration value can be considered as a circulating biomarker of obesity and metabolic syndrome. However, further research is needed to determine whether the theses are correct and to fully understand the pathophysiological functions of ZAG.A study by Yang et al.  in a groIn contrast, the results obtained by Morse et al.  were incLiu et al.  in theirp < 0.05). Reduced ZAG levels were also associated with an increased risk of developing metabolic syndrome . The results of this study suggest that the ratio of serum ZAG to fat mass, may be a future diagnostic biomarker for the diagnosis of metabolic syndrome. Lei et al. reached similar conclusions [In addition, Wang et al.  examinedclusions .2 and 9 obese men (BMI = 34.7 ± 1.2 kg/m2) with a similar lifestyle. Using RT-PCR, they found that the expression of the ZAG gene was reduced by 70% in the subcutaneous tissue of obese individuals compared to slim subjects. Additionally, analysis of the data showed that gene expression positively correlated with serum adiponectin and negatively correlated with plasma leptin levels and waist circumference in obese subjects.In contrast, in the study by Marrades et al. , 18 younp < 0.01). Women with excessive body weight and women with elevated blood glucose levels also exhibited lower blood ZAG levels. It also demonstrated that there was an increase in circulating ZAG after 12 weeks of treatment with either exenatide or metformin (p < 0.01). The study shows that ZAG can be used as an observational indicator in the treatment of PCOS.Overweight and obesity accompany many metabolic diseases, including the polycystic ovary syndrome (PCOS). Studies indicate that ZAG levels are significantly lower in women with PCOS than in healthy women ,96,97. ZUsing genetic profiling, it was found that ZAG transcripts in adipose tissue are reduced in obese women . It has p = 0.00088) and during diet-induced weight loss (p = 0.059). Furthermore, in healthy subjects, ZAG levels were correlated with blood triglyceride levels (p = 0.035), and in obese subjects on a VLCD diet also with LDL-C (p = 0.035 and HDL-C (p = 0.055), but not with TG. The ZAG gene was associated with circulating blood cholesterol levels, which may indicate that ZAG plays a role in its metabolism. This correlation may be due to the role ZAG plays in the body, namely in lipolysis. The researchers suggest that the correlation between ZAG and total cholesterol is the result of increased TG lipolysis, caused by the effects of ZAG.ZAG also potentially affects cholesterol metabolism. A study by Olofsson et al.  showed tEvidence that ZAG is involved in the development of cancer cachexia was presented by Mracek et al. . In cancBoth in vivo and in vitro results show that there is increased hormone-induced lipolysis in patients with cancer cachexia . Rydén eThere are still too few studies to determine the exact pathway through which zinc and ZAG affect lipid metabolism. Both zinc and ZAG are reduced in overweight and obese individuals, which may indicate that a deficiency of these factors indirectly impacts further development of obesity and associated diseases. Zn and ZAG show a negative correlation with body weight, BMI and adipose tissue. Research also indicates that both zinc and ZAG can regulate the lipid profile, thus preventing the development of cardiovascular diseases. In addition, zinc and ZAG have also been shown to affect other adipokines secreted by the adipose tissue. ZAG expression in human adipose tissue was positively associated with adiponectin expression. Serum zinc levels show a negative correlation with inflammatory markers.Summarising the currently available studies, it may be concluded that ZAG and Zn should be more widely used as markers for several biochemical reactions occurring in the human body, both in therapeutic and pathogenetic aspects.


This review aims to investigate the different levels of vitamin D and its role in muscle strength in healthy children and non-athletes. A search conducted in three databases  resulted in 655 articles, which were systematically analyzed and selected based on the following criteria: (a) original cross-sectional studies and clinical trials; (b) healthy children aged 5-11 years; (c) no language restriction or year of publication; and (d) studies that assessed the possible relationship between vitamin D levels and muscle strength. Six studies were included because they met all the inclusion criteria. According to the findings of this review, factors such as sex, skin color, and vitamin D supplementation early in life modulate the levels of vitamin D in the body, and there is a relationship between muscle strength and vitamin D levels. Interestingly, vitamin D supplementation is not always significantly associated with increased muscle grip strength. However, there is a scarcity of studies that aim to analyze the possible effects of different levels of vitamin D on muscle function and neuromuscular variables in physically inactive children and non-athletes without previously diagnosed disease. Further studies are warranted in the future to address the gap in the literature. Serum levels of vitamin D may suffer variability due to genetic and environmental factors related to general metabolism, resulting in changes in its integumentary synthesis and bioavailability ,2. In ad2D) (Vitamin D can increase the capacity to produce strength through the readiness of type II muscle fibers  and favo2D) , the bio2D)  and intr2D) . Thus, vPalacios and Gonzales  reportedThis systematic review adopted the criteria recommended by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) method , which wThe following databases were used to perform our searches: PubMed (MEDLINE), Scopus, and Psycinfo with the following combination of terms contained in the Medical Subject Headings: “Vitamin D” AND “Muscle Strength” OR “Strength, Muscle” and “Child”.Two independent researchers (ABJ and TS) read the study titles and abstracts and, if necessary, read the entire text. In case of disagreement, a third researcher was consulted to establish a consensus. The following inclusion criteria were established: (a) original cross-sectional studies and clinical trials; (b) healthy children aged 5-11 years; (c) no language restriction or year of publication; and (d) studies that assessed the possible relationship between vitamin D levels and muscle strength. Studies that included child athletes, carriers of some diseases, or children receiving maintenance drug therapy were excluded. In addition, the Population Intervention Comparator Outcome strategy was used to select studies with greater specificity.ABJ and TS independently reviewed the selected articles to extract relevant data for the preparation of this review. Conflicts of opinion between the authors were mitigated by a third reviewer. It is important to note that searches performed using electronic databases were performed without the aid of any software.The Joanna Briggs Institute (JBI) tool  was usedA total of 655 titles and 88 abstracts were analyzed. Eighty-two articles were excluded because they did not meet the eligibility criteria or because they were duplicates. A total of 262 studies were excluded because they included unhealthy individuals, which constituted the main cause of exclusion. Four cross-sectional studies and two clinical trials were included. The flowchart for the selection and inclusion of articles is shown in The study participants included in this review were located in different geographic locations, including North America, Europe, and Asia. The age range was 5-11 years. Three studies were identified with insufficient classification of vitamin D levels -24, and It is important to note that vitamin D3 undergoes enzymatic conversion in the liver by vitamin D-25-hydroxylase to produce 25-hydroxyvitamin D (25OHD). 25OHD is the main circulating form of vitamin D in the body.Possible relationships between handgrip strength (HGS) and vitamin D levels were analyzed in all studies, as shown in p=0.96)Filteau et al.  analyzedTrilok-Kumar et al.  studied p=0.005). After supplementation, all children had vitamin D levels ≥50 nmol/L and muscle strength did not differ between groups. Before supplementation, the HGS was 92.18±4.9 N (placebo group), 107.87±5.88 N (10 μg/day) and 106.89±4.90 (20 μg/day). After supplementation, the HGS was 98.06±5.88 N (placebo group), 112.78±5.88 N (10 μg/day) and 109.83±4.90 N (20 μg/day). In this study, there was no difference between the sexes in terms of muscle strength.To determine whether muscle strength is associated with the plasma levels of 25OHD, Mortensen et al.  analyzedp=0.013). However, in girls, there was a significant association , demonstrating an increase in serum 25OHD of 25 nmol/L resulted in an increase in HGS of 2.25 N. It is worth mentioning that the mean HGS value for these children was 82.76±16.96 N.Al-jwadi et al.  found thp=0.010) and Taql genotype “CC” (p=0.038). In addition, three VDR haplotypes were significantly associated with HGS in the dominant and non-dominant hands .Bozsodi et al.  determinThis review investigated scientific literature concerning the relationship between serum vitamin D levels and muscle strength in healthy children. In general, the studies included in this review observed a relationship between muscle strength, measured through HGS, and the serum levels of 25OHD. Variables such as sex, age, and ethnicity can be intervening factors in this relationship. Furthermore, it was found that vitamin D supplementation was not always significantly associated with an increase in HGS, suggesting the need for further studies to consolidate results on this association.It is important to note that none of the studies included in this review (selected according to our inclusion criteria) were executed in countries with a low Human Development Index nor in countries with a tropical climate. Both are important sociogeographic factors that directly influence an individual's serum vitamin D levels, environmentally and with regard to vitamin D synthesis .p=0.256).HGS was used as a predictor of motor performance in healthy children in all studies included in this review. HGS is favorable among muscle function tests because it is simple, non-invasive , and ineA systematic review conducted in 2019 . Humans One of the studies included in this review  presentep<0.001). However, it is necessary to better explain the differences between the sexes to determine the possible relationship between HGS and vitamin D levels from this perspective.Regarding the influence of sex, changes in HGS related to 25OHD levels were observed in children of both sexes. However, only female individuals showed a positive and significant association ,31. In cAccording to our results, supplementation with vitamin D can increase serum 25OHD levels, although this change does not appear to be related to increases in HGS. Studies conducted in children with health impairments have reported the effects of vitamin D supplementation on muscle strength -40 and cAlthough there are discrepancies regarding the association between vitamin D and muscle strength, studies conducted in a population with some motor limitations or in individuals with deficient or insufficient 25OHD levels showed significant positive effects of vitamin D supplementation on neuromuscular abilities ,38,41. IVitamin D deficits in childhood can result in physical and mental health problems in adulthood and old age. One of the many variables that influence serum levels of vitamin D is ethnicity. Some reviews point out that cultural effects may cause vitamin D deficiency, often severe, due to factors such as clothing, beliefs, eating habits, and even the levels of physical activity -47. The Silva ABJ and Carmo TS contributed to the research conception, data collection, interpretation of results, and critical review of the manuscript. Souza APS, Silva MRM, and Fernandes MSS contributed to data analysis and interpretation, manuscript drafting and critical review. Souza VON and Barros WMA contributed to the data collection and critical review of the manuscript.


Buffy coat rLTL and mtDNA content were quantified by monochrome multiplex quantitative polymerase chain reaction. Generalized linear mixed-effects models were used to predict the absolute and percent change in rLTL and mtDNA content after 12 weeks. Iron supplementation was not associated with an absolute or percent change in rLTL after 12 weeks compared with placebo . However, iron supplementation was associated with a smaller absolute and percent increase in mtDNA content after 12 weeks compared with placebo . Thus, daily oral iron supplementation for 12 weeks was associated with altered mitochondrial homeostasis in our study sample. More research is needed to understand the risk of iron exposure and the biological consequences of altered mitochondrial homeostasis in order to inform the safety of the current global supplementation policy.There is limited evidence regarding the potential risk of untargeted iron supplementation, especially among individuals who are iron-replete or have genetic hemoglobinopathies. Excess iron exposure can increase the production of reactive oxygen species, which can lead to cellular damage. We evaluated the effect of daily oral supplementation on relative leukocyte telomere length (rLTL) and blood mitochondrial DNA (mtDNA) content in non-pregnant Cambodian women (18–45 years) who received 60 mg of elemental iron as ferrous sulfate ( Anemia is defined as a hemoglobin concentration <120 g/L in non-pregnant women of reproductive age . In 2011In Cambodia, ~43% of non-pregnant women of reproductive age have anemia . AlthougThere is strong evidence that iron supplementation is effective in treating women who are iron-deplete  . HoweverGlutathione is an abundant antioxidant that plays a critical role in protecting cells against oxidative damage . The ratn = 69 randomly selected women who received iron versus placebo. The results from this pilot study provided the impetus to further investigate markers of cellular damage in the larger study sample (n = 376). Thus, we evaluated the effect of daily oral supplementation with 60 mg of elemental iron for 12 weeks, compared with placebo, on two biomarkers of cellular damage: relative LTL (rLTL) and absolute mtDNA content in n = 376 Cambodian women.First, we conducted a pilot study to measure the GSH/GSSG ratio, an indicator of oxidative stress, in clinicaltrials.gov (NCT-02481375). The complete design, protocol, and methodology have been previously published , p = 0.02) and only adherent women , such that older age was associated with a smaller increase or larger decrease in LTL change. Similarly, baseline rLTL was significantly associated with the adjusted change in rLTL for the models that included all women and only adherent women (p < 0.001). While mtDNA content increased in both groups after 12 weeks, the magnitude of the increase associated with iron supplementation was significantly smaller, compared with placebo, among all women and among only adherent women.p = 1.00 and p = 0.99) or mtDNA content (p = 0.84 and p = 0.71) after 12 weeks. Similarly, the presence of any genetic hemoglobinopathy did not modify the effect of iron supplementation on the adjusted change in rLTL (p = 0.97 and p = 0.95) or mtDNA content (p = 0.09 and p = 0.19) after 12 weeks.Baseline serum ferritin concentration did not significantly modify the effect of iron supplementation on the adjusted change in rLTL (n = 81/191) in the iron group and 58% (n = 108/186) in the placebo group, and iron deficiency based on low ferritin concentrations was 1% (n = 2/190) in the iron group and 26% (n = 48/184) in the placebo group.At 12 weeks, anemia prevalence was 42%  in a model that was adjusted for multiple factors, including age [Similar to the findings described above, some cross-sectional studies have demonstrated a positive association between iron status or exposure and mtDNA content ,52. Datading age . In a reding age  reportedding age .n = 17 had an elevated serum NTBI (≥0.1 μmol/L) after 12 weeks of supplementation, but this did not appear to be associated with iron supplementation (n = 9 were in the Fe group and n = 8 were in the placebo group) [Despite previous cross-sectional studies demonstrating a negative association between iron status or iron supplementation and rLTL ,58,59,60o group) . Howevero group) . We alsoo group) . HoweverHigher doses, durations, and adherence increase iron exposure and can cause more oxidative stress, which may explain why Xu et al.  observedAs our study sample contained a mix of both iron-deplete and iron-replete women at baseline, we assessed the interaction effect between baseline serum ferritin concentration and iron supplementation on both rLTL and mtDNA content at 12 weeks. No interaction was observed, suggesting that the effect of iron supplementation on these outcomes was independent of the presence or absence of iron deficiency at baseline. We similarly observed no interaction effect between the presence of a genetic hemoglobin disorder and iron supplementation on rLTL and mtDNA content. Future studies should also consider these factors as they may impact policy decisions regarding untargeted supplementation regimes.There are no known published studies that have investigated the effect of 60 mg of elemental iron for 12 weeks on the change in rLTL or mtDNA content. The dose and duration of exposure to iron supplements that may lead to the damage of telomeric DNA are not yet clear. The rigorous study design of our randomized controlled trial is better suited to measure changes in rLTL and mtDNA content that are a result of iron supplementation, as compared with cross-sectional studies that can only infer association. Further, the MMqPCR assay used in our study for the rLTL and mtDNA content analyses is a robust high-throughput method that has been validated against other methods of telomere length and mtDNA content measurement . Some liIn conclusion, it is likely that 60 mg/d of elemental iron as ferrous sulfate for 12 weeks does not affect rLTL, but is associated with altered mitochondrial homeostasis. More research is needed to understand the effect of iron supplementation on mitochondrial homeostasis and potential associated health consequences.


Functional neurological disorders (FNDs) are commonly encountered in practice; however, there is a paucity of data in Africa.To identify and describe the clinical profile of patients presenting with FNDs, underlying medical and psychiatric diagnoses and review the investigation and management of these patients.Inkosi Albert Luthuli Central Hospital (IALCH), a tertiary-level hospital in Durban, South Africa.A retrospective chart review and descriptive analysis were performed over a 14-year period (2003–2017) on cases meeting the study criteria.Of 158 subjects, the majority were female (72.8%), had a mean age of 32.8 years, were single (63.3%), unemployed (56.3%) and of black African ethnicity (64.6%). The most common clinical presentation was sensory impairment (57%) followed by weakness (53.2%) and seizures (38.6%). Inconsistency was the most frequent examination finding (16.5%). Medical conditions were identified in half of the study population (51.3%), with hypertension (22.2%) and human immunodeficiency virus (HIV) (17.2%) being most common. Of patients with a psychiatric diagnosis (55.1%), 25.3% had depression. Magnetic resonance imaging (MRI) was the most frequently performed investigation (36.1%). The majority of patients received psychotherapy (72%) and most had not shown improvement (55.3%) at a median follow-up of 2 months, whilst 17% had deteriorated.Functional neurological disorders were most frequently diagnosed in young unmarried females, of black African ethnicity. Family history, personal exposure to a neurological illness and certain socioeconomic factors may be potential risk factors. Sensory impairment was the most common clinical phenotype. Further studies are needed to better understand and manage FNDs in the South African context. Psychiatric terms include conversion disorder, somatisation, dissociative motor disorder, hypochondriasis, factitious disorder, Munchausen syndrome and malingering. All carry different connotations with regard to causation and may affect the patient’s perception of their illness.7‘Functional neurological symptom disorder’ was added to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V) criteria in 2013 and distinguished functional disorders from intentional disorders such as malingering or factitious disorder, although this distinction is debated.8‘Functional neurological symptom disorder’ is a broad term implying a change in function rather than structure and has become a preferred term, as it avoids the insinuation that ‘everything is in the patient’s mind’ and uncertainty as to whether the problem is neurological or psychological.10Functional neurological disorders are commonly encountered in neurology clinics and may cause levels of disability and impaired quality of life similar to multiple sclerosis and Parkinson’s disease.11 and approximately 50% have functional symptoms even if it is not the main complaint.8One-third of neurological outpatients experience symptoms regarded as ‘not at all’ or ‘only somewhat’ explained by disease,Neurologists and non-neurologists alike are often faced with the challenge of inconsistent examination findings, uncertainty as to whether there is co-existent organic pathology and the need to investigate these patients, sometimes at unnecessary cost. In resource-limited settings with high burdens of disease such as South Africa, the need to practise evidence-based, cost-effective medicine is essential.13Although a number of international studies have described the spectrum of FNDs, there is a paucity of data from South Africa and recent literature from Africa. Studies that describe the spectrum of FNDs were either conducted over 50 years ago or if more recent have focussed primarily on psychogenic non-epileptic seizures (PNES) and included relatively small sample sizes.15The aim of this study was to document the experience at a tertiary-level referral centre in Durban, South Africa, and identify the clinical profile, investigation and management of patients with FND presenting to the Inkosi Albert Luthuli Central Hospital (IALCH) over a 14-year period. Inkosi Albert Luthuli Central Hospital serves a population of approximately 18 million people. Patients attending public health facilities in Kwazulu-Natal (KZN) (approximately 79.5% of the population) are mostly of black African ethnicity (90.3%) reflecting local demographics.Addressing the relative lack of local data on this subject may lay the foundation for future research in this important yet neglected field in Southern Africa.A retrospective chart review was conducted on patients presenting to IALCH with a diagnosis of FND between 2003 and 2017. The tenth revision of the International Statistical Classification of Diseases and Related Health Problems (ICD) coding system was used to identify cases. ICD codes F44 – dissociative (conversion disorders), F45 – somatoform disorders, F51 – non-organic sleep disorders and F99 – mental and behavioural disorders were included. Data were extracted by using the Clickview software program, a data extraction program that is able to select patient charts via specific patient numbers according to ICD-10 codes entered. Charts were accessed via the Meditech software system. Patients with an organic neurological disease that may have explained their symptoms and those with insufficient clinical information were excluded. A total of 158 patients were analysed.A data collection tool created by the authors was used to document demographic data, clinical presentation, examination findings, organic disease, psychiatric illness if known or diagnosed during admission, investigations performed, management and follow-up.Descriptive analysis was used to address all the objectives. Continuous variables were summarised by using mean, standard deviation and range, whilst categorical variables were tabulated as frequency and percentage. Categorical variables were analysed by using the Chi-square test, and non-parametric testing was performed for continuous variables. IBM SPSS version 25 was used to analyse the data.The Ethics committee of the Faculty of Health Sciences at the University of Kwazulu-Natal, Durban, South Africa, granted the ethical approval for the study, reference number: BE042/18.During the 14-year study period, 158 subjects with FND were analysed. Patient demographics and characteristics are listed in n = 90/158 (57%), was the most common clinical complaint followed by weakness, n = 84/158 (53.2%), and seizures, n = 61/158 , that reported sensory disturbances. When noted, the sensation was reported to split the midline including vibration sense in six patients. Other sensory complaints were paraesthesias (n = 6), facial numbness (n = 1) and hyperalgesia (n = 1).Hemisensory loss was found in a third of subjects, n = 31/84 (36.9%) of presentations with weakness, followed by hemiparesis, n = 28/84 (33%) (n = 15/28 (53.6%), and when handedness was documented in these patients, all were noted to be right-handed. Thirty-three per cent (n = 3) of the nine patients who presented with quadriparesis reported associated incontinence. This was higher than the percentage of patients who had incontinence in association with other forms of weakness  patients of the total study population complained of weakness. Paraparesis accounted for 84 (33%) . In patin = 33/61 (54%), were more common than focal seizures, n = 22/61 , long duration (n = 5), no postictal phase (n = 1), hyperventilation (n = 1) and high numbers seizures (n = 2).There were 61 patients in whom a diagnosis of psychogenic non-epileptic seizures was documented. Generalised seizures, /61 36%; . Other dn = 12/61) of patients who presented with seizures themselves, a significantly higher proportion than patients without seizures and a positive family history, n = 2/97 . One patient with seizures was noted to live with a known epileptic patient, although not related. Seven patients were admitted for long-term electroencephalographic (EEG) monitoring of seizures, and all studies were normal apart from one documenting a single epileptic seizure amongst many other non-epileptic seizures. Anti-epileptic drugs were stopped in three patients and continued in three patients. Data on treatment discontinuation were not captured for the rest of the sample.A family history of seizures was present in 19.7% , hesitancy (n = 2), appear anxious (n = 2), uncooperative (n = 2), resistant (n = 2) and tearful (n = 1) during examinations.Inconsistency was the commonest clinical finding supportive of FND, 8 16.5%; . Patientn = 87/158). Depression was the commonest psychiatric diagnosis documented overall (n = 18/87) were diagnosed before admission. Of those diagnosed pre-admission, n = 7/18 (38.9%) had depression, n = 6/18 (33.3%) had both anxiety and depression and n = 4/18 (22%) had other diagnoses. When psychiatric diagnoses were made after admission (n = 66), 15 (22.7%) were diagnosed with depression, 1 with anxiety and 14 (21%) with other diagnoses.A psychiatric diagnosis was present in 55% of patients , asthma (n = 5), stroke (n = 4), hypertension (n = 2), multiple sclerosis (n = 1), spinal pathology not specified (n = 1), brain tumour (n = 1), depression (n = 1), visual loss (n = 1) and hearing loss (n = 1)  . Twelve n = 4), work-related problems (n = 1) and sexual orientation (n = 1). Domestic abuse or rape was reported as events preceding the onset of the FND in five patients. Bereavement (n = 2), alcohol abuse (n = 1), incarceration (n = 1) and school failure (n = 1) were noted in others. Trauma related to a motor vehicle accident (n = 3), surgery (n = 1) and a snake bite (n = 1) were also identified as preceding events (Stressors documented included domestic (g events .n = 86/238 (36%), computed tomography scan of the brain (CTB), n = 68/238 (29%), EEG, n = 57/238 (24%), and lumbar punctures (LPs), n = 27/238 (11.3%) (Investigations performed included magnetic resonance imaging (MRI),  (11.3%) . AbnormaElectroencephalographic findings were abnormal in three patients, of which two did not correlate with the patient’s clinical seizure semiology. The other showed an epileptic seizure captured in association with multiple non-epileptic seizures.n = 2/27 (7.4%). One was explained by the patient’s HIV status, and the other had an isolated raised protein of 0.61g/dL with 150 red blood cells noted.Cerebrospinal fluid (CSF) results were abnormal in n = 132/184 (72%), either received or were referred for psychotherapy. The use of antidepressants, n = 32/184 (17.4%), and physiotherapy, n = 20/184 (10.9%), was less common , attended a follow-up appointment. Symptoms had improved in n = 16/47 (34%) of subjects at a median follow-up time of 2 months and had worsened in n = 8/47 (17%), but remained static in the majority of patients 17 but older than in the 2018 Tanzanian study in which almost 50% of patients with FND were below the age of 20.12In keeping with international studies and other African studies, a female predominance was noted.16 reported that most patients with FND were married and well-educated. In that study no association was found between FND and employment status. Our investigation found a predominance of unmarried individuals who were unemployed and of black African ethnicity. Two South African studies reported that most patients (77% and 60%) who presented with PNES were of the white ethnic background; however, these studies analysed patients presenting with PNES only, with one of them limited to patients in a private healthcare setting, which is not accessible to the majority of the local population.19Jacob et al.5 Multiple social stressors including physical and sexual abuse, alcoholism, scholastic failure and bereavement were reported amongst subjects over the study period, which is similar to international data.16As suggested in our study, social circumstances contribute to the development of FNDs and their role is thought to be causative in many cases.20A South African study of pseudoseizures in patients with neurocysticercosis found 17/29 (58%) female patients had experienced sexual or physical abuse and problems related to alcoholism and other socio-economic problems.21 Contributing and causative factors stem from the influences of culture, tradition and religion but the socioeconomic impacts of poverty, alcohol abuse, gun-violence and poor access to legal services are also major role players.22 In addition to the physical and behavioural consequences of GBV, post-traumatic stress disorder (PTSD), major depression and generalised anxiety disorders are documented in victims.22 Domestic abuse or rape was reported in five of our subjects, and psychiatric diagnoses of PTSD, depression and anxiety were also documented. This may aid in understanding why FNDs are more prevalent in the female population in South Africa.Gender-based violence (GBV) in South Africa is amongst the highest in the world.23 Such beliefs are commonly observed in the local population, and many patients may seek the help of traditional healers or ‘sangoma’.24 We identified only two subjects whose family members believed that symptoms were because of witchcraft; however, it is possible that many were not directly interrogated on their beliefs or accessing traditional medicine.Older studies in Africa have documented the influence of cultural beliefs on mental health presentations.27 This corresponds to our finding that a range of psychiatric conditions are prevalent amongst patients with FND, with depression being most common.A high rate of psychiatric comorbidities including depression, anxiety, post-traumatic stress and personality disorders have been documented locally and internationally in patients presenting with PNES.Our study also examined the overlap with organic disease, with hypertension being most commonly followed by HIV, asthma and diabetes mellitus; however, no significant correlations were found. Given the high prevalence of HIV in Southern Africa, this may reflect an under-recognised aspect of the neuro-psychiatric consequences of the HIV epidemic in sub-Saharan Africa. It is possible that the stress of being diagnosed with HIV or HIV-related neurocognitive impairment may contribute to the development of FNDs, but an incidental correlation between the two is also likely because of the high burden of HIV in South Africa.16 Our study did not support the predominance of the PNES phenotype as sensory disturbance was the most common neurological complaint, followed by weakness, seizures and gait dysfunction.In the 2018 Tanzanian case series and 1-year long retrospective review in the United States, the most common FND phenotypes reported were PNES and gait impairment, respectively.28We identified a predominance of paraparesis when patients were weak, hemisensory loss when patients had sensory symptoms and tremor when patients had a movement disorder. Tremor, being one of the most common forms of functional movement disorder, has been documented previously.29 Over half of the hemiparetic subjects in our study had a left-sided weakness.Previous reviews have reported a predilection for the left in up to 60% of patients presenting with functional weakness and sensory symptoms.30 Many of these symptoms were found in our patient sample, with headache being the most commonly documented. Specific diagnoses of IBS, chronic fatigue syndrome and fibromyalgia were not documented; however, pain was a frequently reported symptom suggesting that such functional pain syndromes may have been present.Multiple somatic symptoms such as headache, pain and fatigue are found in patients with FND. Irritable bowel syndrome (IBS), chronic fatigue syndrome, fibromyalgia and chronic pain syndromes are also documented.5 was corroborated by our findings and in particular those who presented with PNES.The impact of family members’ illnesses on phenotypic presentations of FND, or ‘modelling’ as described by Stone et al.,5 A patient may be able to walk but, when examined supine, is unable to lift his or her leg off the bed. Intermittency was documented in 15.8% of our subjects and describes the power that ‘comes and goes’ but is normal with encouragement.5 Variability, whether of frequency, amplitude or distribution was the third most common clinical finding in our study, and not only in relation to movement disorders as is often observed.5 Hoover’s sign, which demonstrates the discrepancy between voluntary and involuntary hip extension, is the only sign found in controlled studies to have a high specificity .32 It was only documented in 5.7% of our subjects. Distractibility, or improvement in abnormal movements when performing tests of mental concentration,5 was also documented infrequently. The ‘hemisensory syndrome’, recognised for over a century, refers to the splitting of sensation down the midline.33 However, in subjects with FND, vibration sense is also discrepant on either side of the sternum or frontal bone despite each vibrating as single units, a distinguishing feature.5 This was observed in six subjects. Not all ‘positive functional signs’ that have been documented in previous studies were assessed in our patients.5 Examining the sensitivity and specificity of particular signs prospectively are a consideration for future studies in South Africa.To distinguish FNDs from organic pathology, ‘positive functional signs’ may be found on examination. Some of these signs include inconsistency, intermittency, variability, Hoover’s sign, distractibility and the ‘hemisensory syndrome’. The commonest signs found in FNDs have not been documented locally. Inconsistency, the most frequently documented sign in our study, refers to discrepancies in findings but does not differentiate whether this is conscious or unconscious.34 A recent systematic review highlighted that individuals with FND and somatic symptom disorders (SSD) might exhibit sensorimotor, prefrontal, striatal-thalamic, paralimbic and limbic structural alterations and therefore may have both a ‘software’ and ‘hardware’ problem.34A number of considerations arise when investigating functional disorders. Investigations are performed to exclude organic pathology but some studies propose neuroimaging to support the diagnosis of FND. This includes the emerging theory of a ‘software problem’, which describes abnormal neurobiological mechanisms (‘software’) despite an intact macroscopic brain structure (‘hardware’).12 As our study reviewed patients attending a tertiary-level hospital with most investigations being readily available, this may not be representative of most hospitals in South Africa where access to specialist care, neuroimaging and neurophysiological studies is very limited, if available at all. This emphasises the need for clinical education on FNDs to ensure that resources are utilised cost-effectively, without compromising the quality of patient care.In resource-limited settings, however, the exclusion of organic pathology overrides proving a pathophysiological basis of FND. For example, the 2018 study in northern Tanzania had access to a single CT scanner and EEG machine with the closest MRI facility being approximately 80 km away.17 the number of investigations carried out and resource utilisation contributed to high costs incurred as a result of FNDs. The authors found that the EEG was the commonest study requested. This is in contrast to the present study, in which MRI was the most frequently performed investigation.In an audit conducted in the United Kingdom by Parry et al.,Cerebrospinal fluid analysis was the least frequently performed investigation analysed in our study and did not aid in diagnosis. Besides the attendant cost, LPs are invasive tests with procedural complications, and in patients with underlying psychiatric illnesses they may precipitate further somatic symptoms. This is another reason, besides cost, why limiting unnecessary investigations in patients with FNDs is crucial.35 These modalities were not analysed in our study as apart from being rarely performed (n = 4/158), their use in the context of FND in South Africa has not been studied.Neurophysiological studies other than the EEG, such as motor-evoked potentials (MEPs) and Bereitschaft potentials, may assist in further defining pathophysiology in FNDs.37Management of FNDs remains challenging, as demonstrated by the high proportion of subjects in our study who showed little or no improvement on follow-up. Whilst data on optimal management strategies are limited, there is evidence that a multidisciplinary team (MDT) approach is effective. This was shown in studies of inpatients with severe and chronic FNDs.38 has been underutilised locally, with only 13.3% of subjects in our study having received physiotherapy. This may have been because of the fact that sensory disturbances were the most common functional symptom documented in our study and clinicians may have been unaware of the benefit of physiotherapy even in these patients. Another consideration is that clinicians themselves did not believe that their patients’ symptoms were ‘real’ and therefore did not warrant physiotherapy.The benefit of rehabilitative physiotherapy, demonstrated in a randomised control study,17The majority of subjects were referred for or received psychotherapy (72%) and a smaller proportion were commenced on antidepressants (17.4%). This represents a higher proportion of patients with FND referred to psychiatric services than in a United Kingdom audit.39Psychotherapy, in particular, cognitive behavioural therapy (CBT) has shown benefit in the management of PNES compared with standard medical care.18 whilst a local public sector study on PNES reported that only 30% of patients received psychotherapy.19 The establishment of similar networks in the public sector would be a major advance in the management of FND and encourage a multidisciplinary approach.Access to psychological support services may vary between the private and public sectors in South Africa. Patients attending an epilepsy-monitoring unit at a private hospital facility were noted to have access to an FDN integrating the work of psychologists, psychiatrists and neurologists,1 An increasing awareness of ‘mind–body’ connections has prompted renewed interest in this area, and relaxation techniques such as ‘mindfulness’ and meditation may hold promise in the future management of functional disorders.Emerging data on the links between emotional states and FNDs have implicated autonomic dysregulation, abnormal limbic motor interactions and abnormal bodily awareness.40Therapeutic sedation has been used with some benefit in a case series although it remains a contentious form of treatment raising ethical concerns.41Newer treatment modalities such as transcutaneous electrical nerve stimulation (TENS) and transmagnetic stimulation (TMS) have not been described in the present study or other local studies, and whether such data from the developed world can be extrapolated to the South African context remains unknown.42The prognosis of patients with FNDs on long-term follow-up appears to be guarded. We found that of the subjects who were followed up in our study, the majority had no improvement in their symptoms and 17% reported deterioration at follow-up. This is in keeping with international data on the outcomes of functional motor disorders and PNES; however, these studies were limited by a lack of validated outcome measures.43 addressed the inconsistency of outcome measures and concluded that few are well validated such as the Functional Movement Disorder Rating Scale (S-FMDRS)44 and the Conversion Disorder Scale (CDS).45 Future studies that apply validated outcome measures will be of benefit to patients and clinicians.A recent review by Pick et al.19 Our study presents the spectrum of FNDs seen at an academic centre in South Africa, and we anticipate that future studies will continue to expand our knowledge and proficiency in this area. As recent studies have suggested, there is a need to eradicate the stigma attached to the diagnosis, to patients and their clinicians and reform the approach taken in managing this condition.7Interest in the complex field of FNDs in South Africa is growing, and as more data on this subject becomes available, current practice will continue to evolve. Lack of training in the identification and management of patients with FNDs is noted to be a barrier to patient care; therefore, raising awareness amongst clinicians is an area of particular concern.13 this study is the first in South Africa to assess the full clinical spectrum of FND, rather than a subset of patients. This is also the largest study conducted on FND in Africa to date. The electronic database available at the hospital provided a reliable, secure data source. The study was conducted in the public healthcare setting, which is accessed by the majority of patients in South Africa and is, therefore, likely to be representative of the general population.Although there have been reports of FND in Africa since the 1950s,This was a retrospective study that did not employ specific outcomes measures, and some patient data were missing. Our study was also limited to a specific geographical area and to a single tertiary referral centre although patients were referred to this facility from throughout the province of KwaZulu-Natal.5 The majority of subjects were female, unmarried, unemployed and of black African ethnicity and the impact of cultural and socioeconomic factors requires further study. Comorbid psychiatric disease and clinical signs such as inconsistency may be useful in establishing the diagnosis of FND. Neuroimaging and neurophysiological studies play a role in the exclusion of organic pathology but should be individualised in a resource-limited setting. The prognosis of patients in this context was poor, but evidence suggests a multidisciplinary approach may offer better outcomes. Further research in Africa is required to expand current knowledge and expertise in this area, reduce stigma and improve both clinician awareness and patient care.Whilst the spectrum of FND is broad and heterogeneous, our study found the most common phenotype to be sensory impairment. A significant proportion of those with PNES had a family history of epilepsy, supporting the theory of ‘modelling’.Abnormal MRI findings included cerebral atrophy, cerebellar atrophy, small vessel disease, sinusitis, periventricular non-specific lesion, chronic lacunas, degenerative spine disease, disc protrusions, old infarcts and an incidental pineal cyst. Only one patient had mesial temporal sclerosis reported on MRI; however, this patient was known with epilepsy and the reason for presenting was a hemiparesis rather than seizures.Computed tomography brain findings included hemiatrophy, cerebellar atrophy, lacunar infarcts, incidental granulomas, calcification, arachnoid cyst, prominent pituitary gland and bilateral basal ganglia hyperintensities.


No gene has garnered more interest than p53 since its discovery over 40 years ago. In the last two decades, thanks to seminal work from Uri Alon and Ghalit Lahav, p53 has defined a truly synergistic topic in the field of mathematical biology, with a rich body of research connecting mathematic endeavour with experimental design and data. In this review we survey and distill the extensive literature of mathematical models of p53. Specifically, we focus on models which seek to reproduce the oscillatory dynamics of p53 in response to DNA damage. We review the standard modelling approaches used in the field categorising them into three types: time delay models, spatial models and coupled negative-positive feedback models, providing sample model equations and simulation results which show clear oscillatory dynamics. We discuss the interplay between mathematics and biology and show how one informs the other; the deep connections between the two disciplines has helped to develop our understanding of this complex gene and paint a picture of its dynamical response. Although yet more is to be elucidated, we offer the current state-of-the-art understanding of p53 response to DNA damage. TP53 gene in humans provide many regulatory functions. Specifically, its role in healthy cells is to suppress changes to a cancerous phenotype by gatekeeping key cellular processes such as apoptosis and cell cycle arrest 92], a co complex , or a hy complex . Bifurcas, as in , appear Spatial models similar to those of Sturrock et al.  and DimiAn alternative approach when developing a p53-Mdm2 network model is to incorporate additional steps or intermediaries, which add additional upregulating pathways. The simplest way in which to couple a positive feedback loop to the negative p53-Mdm2 system is to include a positive feedback loop on p53, a type of model coined as a relaxation oscillator by . This sip53-Mdm2 negative feedback loop with a positive feedbackIf we assume that a physiological delay in the p53-Mdm2 auto-regulatory network is attained through auto-catalytic positive feedback, as shown in This system yields an oscillatory response, for a suitable choice of parameters given in . In FiguZhang et al. study, and compare four different positive feedback mechanisms incorporated into the negative p53-Mdm2 system: Mdm2 as a promoter as well as an antagonist of p53; autocatalytic p53; autocatalytic Mdm2; p53 as a downregulator of Mdm2 . Recall,Other models have incorporated a positive feedback loop on ATM (in this case without Wip1) ,104,105.Another frequent approach is to couple the p53-Mdm2 negative feedback loop to the more complex p53-PTEN-Akt-Mdm2 pathway, which is “globally” positive, see As discussed in The mathematical model proposed by Alon and coauthors has only a few components but it is complicated in the sense that many different relationships are taken into consideration. Moreover, a necessary time lag between p53 activation and p53-dependent induction of Mdm2 is incorporated in the form of a hypothetical intermediary . Neverthbout 6 h , see Figbout 6 h  remains bout 6 h ,112,113.bout 6 h . It is nbout 6 h  and mathbout 6 h ), ratherbout 6 h . Moreovebout 6 h . In Figubout 6 h .As reviewed in the previous sections, most mathematical studies of p53 dynamics in individual cells focus on the underlying mechanisms behind the p53 oscillatory response to DNA damage. They may depend solely on mathematical modelling, or combine mathematical modelling with qualitative/quantitative experimentation. As we have seen, early mathematical models such as ,100,102,Mathematical models may be used to further reveal, so far unknown aspects of p53 signalling, as well as crosstalk between the p53 pathway and pathways of other regulators, activated in response to genotoxic stress. Batchelor et al. investigated p53 signalling in response to UV . They diresponse  without adiation  with thaKonrath and coworkers  combine borators , successNo paper on p53 would be complete without mentioning cancer. Certainly a frequent feature of p53 experiments and mathematical models has been to identify suitable targets for cancer therapy. Indeed, several protocols and p53-based anti-cancer therapies have been developed and already used in clinical trials. These include retrovirus- or adenovirus-mediated gene therapy to restore p53 function lost due to mutations; targeting p53-deficient cells with modified adenoviruses; pharmacological modulation of p53 protein functions; identification and design of small-molecule inhibitors of the Mdm2-p53 interactions such as nutlins or selective Mdm2 inhibitors CGM097 and HDM201 ,118. HowOptimisation of drug administration is perhaps one of the biggest challenges in developing anticancer therapies, for example, prolonged daily administration of some drugs over several days may result in poor tolerability . To deteExtensive PK/PD modelling is used in  to improIn almost all attempts to model p53 intracellular dynamics, many simplifications have to be made, and much biological information has to be excluded. Biomathematicians and mathematical modellers often face the question of what is the “right” information to include in a model? For example, as we have seen in Another contradiction can be found, for example, in the interpretation of ATM-dependent phosphorylation of Mdm2 at Ser395. Experimental and computational works of Batchelor et al. ,82 and MThese two examples and apparent inconsistencies in experimental data point to the value that mathematical modelling can offer this area of research, but pose an important modelling question: simplification or clarification? Which biological processes should be included in the modelling? One can consider all the events hitherto described, even those which appear to oppose each other, and construct a large model (large in the number of parameters). Such an approach is likely to lead to “overfitting”, one can fit whatever experimental data is available with a sufficiently large parameter model, which may lead to false model interpretations and wrong conclusions. Or, one can focus on model reduction and simplification; parameter sensitivity analysis can perhaps point to obvious extraneous model terms, while common generic network motifs can be incorporated . Indeed Toettcher et al. lead by example, by introducing what they call a “synthetic-natural oscillator”; a model in which core aspects of the network are modelled in full while periphery interactions are distilled into artificial terms . The issIt is perhaps fair to say that the technologies to study oscillations in single cells remain quite limited and this simple fact is why we still do not have the full picture of the dynamical behaviour of p53 under normal versus stressed conditions. Since there are several ways one can view oscillations, mathematics can certainly help to fill some of the gap. It is perhaps important to identify as many oscillators as possible in a particular pathway, to try to understand, from both experimental and mathematical points of view, if and how these different factors interact with each other, what their individual functions in the oscillations are, and how they communicate. Do they have the same target, or do they act on several targets in the same pathway? It is conjectured that there must be some limitations to the cellular repertoire, in terms of which mechanisms are in place to generate an oscillating feedback system. How many of these are required as a minimum to achieve a tightly regulated cell biological response?“The Functional Landscape of p53” was to offer a snapshot of the different mathematical mechanisms, which provoke oscillatory p53 dynamics, as well as commenting on the interplay between mathematical modelling and experiments in this lively field. Although every effort has been made to provide a comprehensive survey of the literature, the topic of p53 has garnered much interest from experimentalists and mathematicians alike. For additional reviews of the extensive literature please see [Over the last 20 years a wealth of study into the dynamics of p53 has permeated the literature. The purpose of this review for this special edition ease see ,132,133.


Skin cancers are the most common types of cancer worldwide, and their incidence is increasing. Epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, and it is associated with poor prognosis in the advanced stage. The most important risk factor for cSCC is long-term exposure to solar ultraviolet radiation, which induces oncogenic mutations in epidermal keratinocytes. The most common mutations are inactivating mutations in tumor suppressor p53, which result in accumulation of additional mutations. Recently, the role of p53 in the progression and invasion of cSCC has also been elucidated. In this review we will discuss the role of p53 in development of cSCC and as a potential new therapeutic target in advanced cSCC.TP53 gene and inactivation of the tumor suppressor function of the tumor protein 53 gene (TP53) in epidermal keratinocytes, which then leads to accumulation of additional oncogenic mutations. Additional genomic and proteomic alterations are required for the progression of premalignant lesion, actinic keratosis, to invasive and metastatic cSCC. Recently, the role of p53 in the invasion of cSCC has also been elucidated. In this review, the role of p53 in the progression of cSCC and as potential new therapeutic target for cSCC will be discussed. Skin cancers are the most common types of cancer worldwide, and their incidence is increasing. Melanoma, basal cell carcinoma (BCC), and cutaneous squamous cell carcinoma (cSCC) are the three major types of skin cancer. Melanoma originates from melanocytes, whereas BCC and cSCC originate from epidermal keratinocytes and are therefore called keratinocyte carcinomas. Chronic exposure to ultraviolet radiation (UVR) is a common risk factor for skin cancers, but they differ with respect to oncogenic mutational profiles and alterations in cellular signaling pathways. cSCC is the most common metastatic skin cancer, and it is associated with poor prognosis in the advanced stage. An important early event in cSCC development is mutation of the  Skin cancers are the most common types of cancer worldwide, and their incidence is increasing. Melanoma, basal cell carcinoma (BCC), and cutaneous squamous cell carcinoma (cSCC) are the three major types of skin cancer. Melanoma originates from melanocytes, whereas BCC and cSCC originate from epidermal keratinocytes. BCC is derived from basal cells of the interfollicular epidermal layer, and cSCC is derived from keratinocytes of the interfollicular epidermal layer and hair follicle stem cells . ChronicNOTCH1 . Cli. Cli1]. Only a portion of AKs will progress to invasive cSCC ,28. The TP53 in epidermal keratinocytes is an early event in cSCC development  and p14(ARF), are also common in cSCC [CDKN2A occurs at later stage in the progression from AK to cSCC, since it is not mutated in the sun-exposed normal skin  genes in cSCC include nd ERBB4 ,48. Sustnd ERBB4   can grow over wild-type cells to colonize normal epidermis, but the epidermis can adapt to the mutant clones and revert the expansion of the mutant cells over time [It has been shown that p53 mutations arise very early in mouse skin after UVB exposure, and that the growth of the p53-mutant keratinocytes is driven by UVB ,89. Intever time . Short-tver time . What dever time . In addiver time . TP53 and NOTCH1/2 [TP53 is mutated three times more frequently in the normal skin of patients with a high skin cancer burden compared to the low-burden group, indicating that elevated accumulation of UV-signature mutations is associated with an increased burden of cSCC [Normal sun-exposed skin adjacent to a cSCC harbors a high mutation burden, including the UV-targeted genes NOTCH1/2 . The oveNOTCH1/2 . Particu of cSCC .+/−;p73+/−) drives spontaneous cSCC development in mice, indicating that p73 may play a role in cSCC development [p53 shares a high degree of structural and functional homology with two of its ancestral genes, p63 and p73 . They arelopment . The ΔNpelopment .TP63 gene has been reported in cSCCs, and given the substantially higher expression of the ΔNp63 compared to TAp63 in normal epidermis, the oncogenic function of p63 is primarily dependent on the overexpressed ΔNp63 isoform in the skin [A1AT), which is upregulated through the mut53–p63 complex, promotes tumor invasion by inducing the expression of several epithelial–mesenchymal transition (EMT) markers [p63 and p73 are rarely mutated in cancer, but the balance between their different isoforms can be critical for the cell fate and cancer development ,95. For the skin . The ΔNpthe skin . Moreovethe skin . The strthe skin ,95. In athe skin . The co-the skin . One suc markers .TP53 gene are early events in skin carcinogenesis, and they predispose skin cells to further genomic instability. In accordance with this, several studies show remarkably high overall mutational burden in human cSCCs, which occur mostly at older age [Mutations in the lder age ,5,6,7. TTP53 gene in cSCC tumors is very high, ranging from about 50% in primary cSCCs to nearly 95% in aggressive tumors [TP53 gene, but not all mutations are equal. As in many other human cancers, also in cSCCs the missense mutations in TP53 gene are frequently found in the conserved p53 DNA-binding domain  oncogenic activities, such as increased pro-growth signaling, invasiveness, and tumor metastasis B 104,10,106. A dR248W contact mutant is commonly detected in cSCCs  [R175H exhibits GOF activity in many cancer types. For example, in HNSCC cells, p53R175H drives the expression of oncogenic transcription factor FOXM1, which is upregulated in human oral premalignant and HNSCC tissues [R175H has been shown to interact with other p53 family members, p63 and p73, and inhibit their transcriptional program to promote cell invasion [R175H drives cSCC development in mice, when specifically expressed in the epidermis [In addition to the contact mutant p53 , missense mutations in the (HNSCCs) , and it (HNSCCs) , althoug tissues . Mutant invasion ,115,116.pidermis . This p5pidermis ,119. TP53 gene, a significant fraction of human cancers carry TP53 nonsense mutations, which introduce a premature stop-codon [In addition to the missense mutations in the op-codon . This wiop-codon . These nop-codon . Expecteop-codon ,125,126.op-codon . Not allop-codon ,129. Intop-codon . In addiop-codon . In accoIn summary, distinct p53 mutants possess a plethora of functional mechanisms in which they can regulate several hallmarks of cancer, from activation of cell proliferation to promotion of invasion and metastasis. The biological consequence of distinct p53 mutants is very context dependent. Moreover, the recent evidence of the cancer-associated p53 isoforms, particularly the truncated ones which can escape degradation, indicates that the loss of function or nonsense p53 mutants should be regarded as an additional subgroup of GOF p53 mutants. Yet, the existence and potential function of different p53 isoforms in the process of skin carcinogenesis is not known.TP53 plays a significant role in cSCC development. Mutant p53 is stable and accumulates in the nucleus, and a strong p53 immunopositivity is a widely used characteristic to detect mutated p53 in tumor tissues [TP53 mutations are frequently detected in normal sun-exposed skin and AKs, increased p53 immunopositivity in AKs is associated with increasing severity of dysplasia, and with progression of AK to cSCC [TP53 mutations present an early indication of UV damage, and increased TP53 mutational frequency is associated with potential cSCC development. However, there is also evidence that UV-induced mutation and inactivation of NOTCH1 may precede mutation of TP53 [Mutated  tissues ,130. Con tissues ,102. The tissues . While p to cSCC ,89,131.  of TP53 .The protective role of tumor suppressor p53 in skin cancer has been shown in several studies. Loss of p53 accelerates skin tumor formation after UV irradiation , and losCDKN2A gene encodes two important tumor suppressors, p16(INK4a) and p14(ARF), which are frequently inactivated in cSCC , which constitute an essential layer of gene regulation ,138. MicNORAD (noncoding RNA activated by DNA damage), are direct transcriptional targets of p53 [MiRNAs are evolutionarily conserved, single-stranded ncRNAs ~22 nucleotides in length. They inhibit gene expression specifically by binding to the 3’-untranslated region of the target mRNA in the cytoplasm, resulting in translational repression or degradation of target mRNA . Compares of p53 ,143.MALAT1 (metastasis associated lung adenocarcinoma transcript 1), which is commonly upregulated in cancer, has been shown to interact with mutated p53 in breast cancer [MALAT1 to mutant p53-ID4 protein complex, delocalizing MALAT1 from nuclear speckles and favoring its association with chromatin [As discussed above, the tumor-suppressive function of p53 is frequently lost in cancer, particularly in skin cancers, and mutated p53 can acquire oncogenic GOF activity. In addition to the proteins that cooperate with mutated p53 to transactivate specific target genes in cancer, also ncRNAs have been shown to participate in the pathogenic signaling via p53 mutants . As an et cancer . Here, ohromatin . This wihromatin .TPX2 (TPX2 microtubule nucleation factor) is upregulated [TP53 mutation and aggressive clinical behavior in breast cancer, supporting a TPX2-p53 regulatory circuit [While the role of ncRNAs in cSCC progression is emerging ,146, onlegulated . High nu circuit . In cSCC circuit . Another circuit .PRECSIT (p53 regulated carcinoma-associated STAT3 activating long intergenic non-protein coding transcript) is a recently characterized lncRNA with a tumorigenic function in cSCC [TP53 can cause premature p53 translation termination, leading to undetected p53 protein expression. Here, PRECSIT was shown to be upregulated in cSCC, and its high expression was specifically associated with loss of p53 in cSCC in vivo [PRECSIT expression in cSCC progression. In accordance with this, delivery of a wild-type p53 into cSCC cells, which harbor mutated p53 led to downregulation of PRECSIT expression, indicating that its expression is suppressed by p53. PRECSIT was further shown to activate STAT3-signalling and expression of MMPs and invasion of cSCC cells [MiRNA regulation is very context-dependent, and certain miRNAs are specifically regulated by mutant p53. For example, miR-1246 is induced by mutant p53 in colon cancer, and miR-34 is suppressed by mutant p53 in lung cancer . Thus, i in cSCC   has been shown to restore the proper folding and transcriptional activity of p53 mutant inducing apoptosis in the p53ft model . In addiell line . Howeverr cSCCIS ,113.Delivery of the p53 gene directly into tumor cells is one option to target p53 mutations, and wt-p53 can be transferred to cells by viral vector in combination with cancer treatments. Combining chemotherapy, immunotherapy, or radiation therapy with the p53 gene therapy may increase the efficacy of tumor cell targeted therapies. For example, in HNSCCs, intra-tumoral p53 gene delivery is being studied. In phase 2 multi-center open label clinical trial adenoviral p53 (Ad-p53) gene therapy is administered intratumorally in combination with immune checkpoint PD-1 and PD-L1 inhibitors in patients with recurrent or metastatic HNSCC and other solid tumors approved for anti-PD-1 or anti-PD-L1 therapy . AnotherIn summary, p53 targeting is an interesting area in drug development, but the efficacy of p53 targeted therapies has not yet been tested in advanced and metastatic cSCCs. It is conceivable that drugs targeting several mutants of p53 are needed, and that p53-targeting drugs would be used in combination with surgical excision, immunomodulators, radiation therapy, or chemotherapies. However, given the important role of p53 mutations in the progression of cSCC, it is expected that p53-targeted therapies could be useful already at an earlier stage of cSCC development, in addition to treatment of advanced and metastatic cSCC.Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, and it is associated with poor prognosis in the advanced stage. The mutation rate of cSCC is one of the highest among the malignant tumors, and the majority of mutations found in cSCC are UV-induced. An important early event in cSCC development is mutation and inhibition of the wild-type function of tumor suppressor p53. This leads to the accumulation of additional oncogenic mutations. It has become evident that additional alterations, for example in non-coding RNAs, are required for the progression of premalignant lesion and actinic keratosis to invasive and metastatic cSCC. In addition, the role of p53 in the invasion of cSCC has also been elucidated. Together, these observations suggest mutant p53 as a putative target in both at the early stage of cSCC progression, as well as in advanced and metastatic stages. It is possible that multiple p53 targeted therapeutic approaches under development may be feasible in the treatment of cSCC at different stages of tumor progression.