The treatments lasted for 12 weeks. Obese animals showed increase in body weight, visceral fat mass, insulin and triglyceride-glucose index and a reduction in insulin sensitivity. In addition, obese animals also showed increase in plasma/hypothalamic and adipose pyruvate dehydrogenase kinase-4, lactate-pyruvate ratio, malondialdehyde, γ-glutamyl transferase, and a decrease in glucose-6-phosphate dehydrogenase, glutathione, nitric oxide and PPAR-γ. HFD also elevated plasma/hypothalamic lipid and decreased adipose lipid profile, increased hypothalamic and adipose tumor necrosis factor-α, interleukin-6 and histone deacetylase (HDAC), and elevated plasma/adipose leptin. These alterations were reversed by concomitant administration of acetate. The present results demonstrate that obesity is characterized by BAMED, which is accompanied by altered HDAC/PPAR-γ. The results in addition suggest that acetate, an HDAC inhibitor rescues BAMED with consequent normalization of body weight and visceral fat mass by modulation of PPAR-γ and suppression of oxidative stress.We investigated the hypothesis that acetate ameliorates brain-adipose metabolic dysfunction (BAMED) in high fat diet (HFD)-induced obesity, possibly by modulation of peroxisome proliferator-activated receptor-γ (PPAR-γ). Ten-week-old male Wistar rats were randomly assigned into four groups (n = 6/group): Control, acetate and obese with or without acetate groups received vehicle (distilled water;  Based on the current prevalence, over 38% of the global population is estimated to be overweight or obese by 20303. Obesity was initially evident to be a problem of the developed countries and even as it continues to rise in many of these countries, its prevalence continues to trend upward in developing countries, including among the children and adolescents4. It is usually characterized with excessive adiposity and any of the following: insulin resistance, hyperglycemia and overweight5. Obesity constitutes a risk factor for a number of chronic diseases including type 2 diabetes mellitus (T2DM), cardiovascular diseases (CVDs), depression, renal disease, liver disease, reproductive dysfunction and cancer, and has adverse effects on overall health as well as global public health7. In fact, the economic and psychosocial burden of obesity coupled with its comorbidities is rapidly escalating.Obesity is a complex multifactorial metabolic disease, affecting more than 30% of global population as overweight or obese, and attributing to 8% of the annual worldwide mortality9. Previous studies reported that genetics influence the body mass index (BMI) which is a commonly used indicator of obesity and accounts for energy requirements, fuel utilization and muscle metabolic characteristics12, however, the current obesity epidemic has been shown to be principally driven by behavioral and environmental factors1. Sedentary lifestyle and consumption of high energy diet are the most common contributors to the energy imbalance that causes overweight and excessive fat deposition9. Nevertheless, the pathogenic processes underlying obesity are multifactorial and still under elucidation which posit a barrier for designing effective strategies towards prevention and management.The cause of obesity has been linked to behavioral , environmental and genetic factors13. Physiologically, leptin is secreted by adipocytes in proportion to total fat mass14 and regulates energy balance by reducing energy intake or increasing energy expenditure13. It reduces energy intake by decreasing food or caloric intake through appetite inhibition in the hypothalamus, thereby preventing excess body weight15. It also increases energy expenditure by elevating lipid-glucose utilization, thus limiting excess energy storage or excessive fat deposition in the adipose tissue16. The regulatory functions of leptin occur by binding to hypothalamic leptin receptors, which signals to downstream effectors through the Janus kinase/signal transducers and activators of transcription factors (JAK/STAT) signaling pathway13. On the other hand, impaired leptin tolerance elicits metabolic signals that promote energy storage inform of fat deposition and caloric intake, which are crucial features of obesity, thus associating obesity with high leptin concentration15. Earlier studies have demonstrated leptin resistance in experimental rodents fed with high fat diet (HFD)17, suggesting that HFD consumption limits the action of leptin in the regulation of energy balance regardless of its concentration. Similarly, hyperleptinemia could also induce or aggravate leptin insensitivity by upregulating the suppressor of cytokine signalling-3 (SOCS-3) with consequent blockade of signal transduction of leptin18.However, a number of previous investigations have documented leptin resistance as a critical factor in the development of obesity21. Study by Thaler et al., reported the evidence of hypothalamic inflammatory signaling in rodents within 1 to 3 days of HFD exposure, prior to substantial weight gain22, suggesting the possible involvement of hypothalamic inflammation in the pathogenesis of obesity, unlike inflammation in the peripheral tissues which develops as a consequence of obesity. This study added that both reactive gliosis and markers suggestive of neuron injury were evident in the hypothalamic arcuate nucleus of rodents within the first week of HFD exposure and extends to mediobasal hypothalamus with chronic exposure22. Nevertheless, evidence reveals hypothalamic lesion in obese humans as assessed by magnetic resonance imaging23, suggesting that obesity is critically associated with hypothalamic injury in both humans and experimental rodents. Similarly, more investigations have demonstrated elevated level of circulating pro-inflammatory cytokines, proteases and growth factors derived from adipose tissue of obese rodents, which possibly contributes to an increased hypothalamic inflammatory signaling that mediates leptin resistance and weight gain21. However, the cellular interactions underlying the brain-adipose inflammatory response in the development of obesity is not clear.In addition, the development of obesity which is characterized by leptin resistance has been associated with inflammation both in peripheral tissues including adipose tissue and in hypothalamic areas critical for energy homeostasis25. PPARS are activated or repressed in response to regulatory networks of genes controlling various physiological processes that involve inflammation, adipogenesis, lipid-glucose metabolism and insulin sensitivity27. Defects in PPARs have been linked to metabolic stress and related syndrome, including the impaired adipose tissue remodeling and function28, which are detrimental during nutrient deprivation when adipose tissue remodeling is required for cellular survival. Earlier studies have demonstrated PPARs as lipid sensors that are capable of improving energy homeostasis when optimally activated29. Nonetheless, considering the therapeutic potential of PPARS activation in metabolic stress and related disorders, it would be of clinical relevance and public health interest to investigate the possible involvement of PPARs, especially PPAR-γ in brain-adipose inflammatory events underlying obesity.Peroxisome proliferator-activated receptor-γ (PPAR- γ) is among the three isoforms of PPARS, which are nuclear hormone receptors that exert transcriptional control on genes housing PPAR-responsive regulatory elements and heterodimerize with retinoid X receptors31. Post-translational modification of proteins involving acetylation or deacetylation are governed by histone acetyltransferases (HAT) and histone deacetylases (HDACs) which transfers acetyl group to histone proteins and causes reversal of acetylation respectively. Evidence exists that the PPAR-γ gene is not only genetically regulated, but also regulated by epigenetic modulation, including histone modification and its regulatory enzymes such as HAT and HDACs33. Similarly, the functional roles of epigenetic enzymes, including HDACs in the PPAR-γ transcriptional pathway have been suggested35. In addition, certain post-translational modifications could also be mediated by pro-inflammatory and pro-oxidant species leading to suppression of gene expression36. Therefore, therapies targeting HDACs might be a potential promoter of PPAR-γ with possible attenuation of visceral adiposity, and in addition be a novel safer and effective intervention for the management of obesity. Acetate is a short chain fatty acid (SCFA) metabolite produced from distal gut microbiota that has been attributed to a wide range of protective health benefits37. Acetate is known to regulate metabolic activities of cells by inhibiting HDAC and/or activating G-protein coupled receptors signaling. It improves intestinal mucosa integrity/immunity, enhance insulin sensitivity and exert anti-inflammatory effects. Earlier studies demonstrated that oral supplementation with acetate exerts recovery from hepatic lipotoxicity39, neuroinflammation40, and hypertension41. However, the effects of acetate on disrupted brain-adipose metabolic link and/or brain-adipose inflammatory events underlying obesity was not known. The present study was therefore designed to test the hypothesis that SCFAs, acetate ameliorates defective brain-adipose metabolic network associated with high fat diet (HFD)-induced obesity in male Wistar rats, possibly by modulation of PPAR-γ.Epigenetic modulation of key metabolic enzymes contributes to the pathogenesis of obesityThere was a significant increase (p < 0.05) in body weight and visceral fat mass in obese animals compared with control animals. However, the administration of acetate to obese plus acetate (OBS + ACT) group significantly reduced (p < 0.05) the body weight and visceral fat mass compared to the untreated obese animals and no significant difference when compared to the control group Fig. A,B. AlthThough the fasting blood glucose remained unchanged (p < 0.05) but a significant decrease in glucose tolerance and insulin sensitivity were observed in obese animals compared with control animals and these were significantly increased (p < 0.05) following the administration of acetate to OBS + ACT group compared to the untreated obese group and no significant difference when compared to the control group. In addition, there was a significant increase (p < 0.05) in fasting plasma insulin and triglyceride-glucose (TyG) ratio in obese animals compared with control animals and these were normalized following the administration of acetate to OBS + ACT group compared to the untreated obese group Fig. .Figure 2There was a significant decrease (p < 0.05) in plasma, hypothalamic and adipose glucose 6 phosphate dehydrogenase (G6PD) with corresponding increase in pyruvate dehydrogenase kinase-4 (PDK4) in obese animals compared with control animals. However, administration of acetate significantly increased (p < 0.05) the plasma, hypothalamic and adipose G6PD with corresponding decrease in PDK4 in OBS + ACT group compared to the untreated obese group and no significant difference when compared to the control group Fig. .Figure 3There was a significant increase (p < 0.05) in plasma, hypothalamic and adipose lactate-pyruvate ratio with corresponding increase in GGT activity in obese animals compared with control group. Nevertheless, administration of acetate significantly decreased (p < 0.05) the plasma, hypothalamic and adipose lactate-pyruvate ratio and GGT activity in OBS + ACT group compared to the untreated obese animals and no significant difference when compared to the control group. Nonetheless, hypothalamic lactate-pyruvate ratio was significantly higher OBS + ACT group when compared to the control group Fig. .Figure 4There was a significant increase p < 0.05) in plasma triglyceride/high-density lipoprotein cholesterol (TG/HDLc) and total cholesterol/HDLc (TC/HDLc) and hypothalamic TG and TC in obese animals compared with control animals. However, administration of acetate attenuated dyslipidemia and hypothalamic lipid accumulation in OBS + ACT group compared to the untreated obese group. Besides, adipose TG and TC decreased significantly (p < 0.05) in obese animals compared with control animals, which were attenuated following the administration of acetate in OBS + ACT group compared to the untreated obese group and no significant difference when compared to the control group  in plasma, hypothalamic and adipose malondialdehyde (MDA) and a decrease in glutathione (GSH) concentration in obese animals compared with control animals. Nevertheless, administration of acetate significantly decreased plasma, hypothalamic and adipose MDA with corresponding increase in GSH concentration in OBS + ACT group compared to the untreated obese group and no significant difference when compared to the control group Fig. .Figure 6There was a significant increase (p < 0.05) in hypothalamic and adipose tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in obese animals compared with control animals and these alterations were reversed following the administration of acetate to OBS + ACT group compared to the untreated obese group and no significant difference when compared to the control group Fig. .Figure 7There was a significant decrease (p < 0.05) in plasma, hypothalamic and adipose nitric oxide concentration in obese animals compared with control animals. However, administration of acetate significantly increased (p < 0.05) the plasma, hypothalamic and adipose nitric oxide (NO) concentration in OBS + ACT group compared to the untreated obese group and no significant difference when compared to the control group Fig. .Figure 8There was a significant increase (p < 0.05) in plasma and adipose leptin concentration in obese animals compared with control animals. However, administration of acetate normalized the circulating and adipose leptin concentration in OBS + ACT group compared to the untreated obese group. Nevertheless, plasma ghrelin concentration was not altered in obese group compared to the control group Fig. .Figure 9The hypothalamic and adipose levels of HDAC significantly increased (p < 0.05) in obese animals compared with control animals. Nevertheless, administration of acetate significantly decreased (p < 0.05) the hypothalamic and adipose HDAC levels in OBS + ACT group compared to the untreated obese group and no significant difference when compared to the control group Fig. .Figure 1The plasma, hypothalamic and adipose levels of PPAR-γ significantly increased (p < 0.05) in obese animals compared with control animals. Nonetheless, administration of acetate significantly increased (p < 0.05) the plasma, hypothalamic and adipose levels of PPAR-γ in OBS + ACT group compared to the untreated obese group and no significant difference when compared to the control group Fig. .Figure 1The key finding from the present study is that acetate as HDAC inhibitor attenuates defective hypothalamic-adipose metabolic network in HFD-induced obesity and this beneficial effect of acetate is accompanied by elevated PPAR-γ and suppressed oxidative stress. The results of the study in general showed that HFD induces obesity, which is characterized with increased body weight, visceral fat mass, fasting insulin and triglyceride-glucose index and decreased insulin sensitivity with unaltered blood glucose when compared to the control group. In addition, obese animals also showed increased plasma, hypothalamic and adipose PDK4, lactate-pyruvate ratio, GGT, lipid peroxidation (MDA) and decreased antioxidant capacity (G6PD and GSH), NO and PPAR-γ, elevated plasma and hypothalamic lipid and decreased adipose lipid profile, increased hypothalamic and adipose inflammation  and HDAC with corresponding increase in adipose and circulating leptin compared to the control group. However, administration of acetate reversed these alterations with consequent normalization of body weight and visceral fat mass by suppression of oxidative stress and upregulation of PPAR-γ Fig. .Figure 19. The present results that high fat diet induced obesity with evidence of increased body weight gain and visceral adiposity when compared to the control group show consistency with earlier observations. As extension, the results showed that the present model of obesity is associated with insulin resistance and hyperinsulinemia as revealed with elevated TyG/reduced insulin sensitivity and increased fasting insulin concentration respectively. Although the fasting blood glucose was not affected when compared to control group despite the impaired glucose tolerance observed in obese animals. The findings here suggest that in addition to excess body weight or visceral adiposity, the obese animals are characterized with disrupted glucose homeostasis regardless of the normal blood glucose level that might have resulted from the compensatory hyperinsulinemia. Besides, the findings also imply that the obese animals are pre-diabetic, which is a dysmetabolic state of glucose level that is primarily characterized by impaired glucose tolerance, thus validating earlier observations that obesity constitutes an independent risk factor to T2DM and attendant CVDs7. Nevertheless, the brain weight of obese animals was not affected when compared to the control group, although the development of obesity has been associated with changes in organ weight, particularly, the liver, kidney and heart43. However, other studies have reported that changes in organ weight is dependent on the etiology of obesity45.Obesity has been well characterized by excess body weight and visceral fat mass with or without insulin resistance in both experimental rodents and humans47 which is corroborated by dysregulated glucoregulatory enzymes, especially G6PD and PDK4, an isoform of PDK. Glucose 6 phosphate dehydrogenase and PDK4 are critical metabolic enzymes that are involved in energy production inform of ATP either through glycolysis or pentose phosphate pathway (PPP)49. Glucose 6 phosphate dehydrogenase is the first step and rate-limiting enzyme in the oxidative stage of PPP, which results in production of ribose-5-phosphate49, while PDK, including PDK4 inhibits pyruvate dehydrogenase, an enzyme that regulates the oxidative metabolism of glucose through pyruvate dehydrogenase complex that promotes the conversion of pyruvate to acetyl coA51. In the present study, there was a significant decrease in plasma, hypothalamic and adipose G6PD with corresponding increase in PDK4 in obese animals compared to control group. These results therefore indicate that glucose metabolism or utilization is impaired in obese animals, which seems similar to previous studies, including studies from our laboratory that reported elevated PDK in animal models of obesity53 and in obese humans54. In addition, impaired glucose metabolism or utilization for ATP production favors conversion of pyruvate into lactate as revealed by elevated plasma, hypothalamic and adipose lactate-pyruvate ratio in obese animals compared with control group. Lactate-pyruvate ratio is a marker of cytosolic redox status55, and its increased level in obese animals possibly triggered hypothalamic and adipose tissue ischemia which moved both tissues toward proinflammatory phenotypes (elevated TNF-α and IL-6) and oxidative stress (increased MDA and decreased GSH) and aggravated by NO depletion. These perhaps led to tissue injuries as biochemically characterized by elevated circulating, hypothalamic and adipose GGT activity in obese animals compared to the control group. Therefore, the present findings suggest that impaired glucose metabolism or utilization at least in part promotes hypothalamic-adipose tissue injuries in obese animals.Disruption of glucose homeostasis observed in obese animals is a reflection of impaired glucose metabolism56. This possibly caused hypothalamic metabolic stress or lipotoxicity that triggered hypothalamic inflammatory response as revealed by elevated levels of hypothalamic TNF-α and IL-6 in obese animals compared to the control group. The observation here is in consonance with previous studies that reported the evidence of hypothalamic inflammatory signaling in rodents within 1 to 3 days of HFD exposure, prior to substantial weight gain due to energy imbalance22. Besides, hypothalamic metabolic stress could also be responsible for increased lipid peroxidation (MDA) with corresponding decrease in GSH, which possibly triggered hypothalamic oxidative stress with consequent injuries as revealed by elevated GGT activity in obese animals compared to the control group. On the other hand, adipose TG and TC significantly decreased in obese animals compared to control group. This observation is consistent with earlier studies, including a recent study from our laboratory, which reported that insulin-resistant adipose tissue is characterized with increased white visceral adiposity, that remodels and expands causing adipose dysfunction with corresponding decrease in adipose lipids including TG and FFA58 due to increased lipolysis. This further aggravates systemic and peripheral insulin resistance that underlie complications associated with obesity7. In addition, previous studies reported that HFD provoked insulin resistance-driven adipose remodeling, which often overwhelmed in chronic condition causing numerous macrophages invasion surrounding the dead adipocytes, thus triggering a crosstalk between the lipid, macrophages and toll like receptors located on the macrophages with consequent induction of proinflammatory cytokines60 as validated in the present study with elevated levels of circulating and adipose TNF-α and IL-6 in obese animals compared to the control group. The elevated levels of TNF-α and IL-6 in the adipose tissue are due to the enlargement of white adipose tissue (increased adiposity), which characterized obese animals causing the recruitment and activation of immune cell subsets in the white adipose tissue, which systemically induces the low-grade inflammation that in turns aggravate systemic and peripheral insulin resistance, contributing to the complications of obesity as earlier reported60. Therefore, the results herein imply that hypothalamic-adipose-lipid dysmetabolism drives hypothalamic/adipose inflammatory response that characterized obesity.Furthermore, the development of insulin resistance in obese animal reduced sensitivity of metabolic tissues including adipose tissue to insulin action as revealed by QUICKI, this possibly increased lipolysis with corresponding increase in circulating lipids such as TG/HDLc and TC/HDLc resulting in excess lipid influx into the non-metabolic tissues, including hypothalamic tissue with correspondent elevated TG and TC compared to the control group. The deposition of excess lipid in the hypothalamus has been earlier reported to reduce insulin sensitivity with corresponding decrease in hypothalamic insulin-mediated glucose uptake62. However, hypothalamic inflammatory response reduced the sensitivity of the hypothalamus to leptin (leptin resistance) leading to an increase in the plasma concentration of leptin in obese animals compared to control group. Hence the present results are consistent with earlier investigations by Thaler and Schwartz and Bittencourt et al., who demonstrated hyperleptinemia with consequent energy dysregulation in obese rodents that suffer hypothalamic lesions predominant with inflammation21. In addition, the results of the present study did not show significant alteration in the circulating level of ghrelin in obese animals compared to the control group, though earlier studies have shown inconsistent results in the status of ghrelin in obese individuals65. Therefore, the findings from the present data suggest that obesity may be independent of ghrelin concentration but is characterized by hyperleptinemia, which is attributable to increased visceral adiposity and leptin resistance invoked by hypothalamic inflammatory mediators.Besides, increase in visceral adipocyte possibly contributed to elevated leptin production in obese animals compared with control group. This observation is in consonance with earlier studies that reported a positive correlation between leptin and adipocytes in obese humans28, a pathological process that also occurs during adipose expansion and often results in nitric oxide deficiency and elevated inflammatory mediators as observed in obese animals compared to control group. Besides, earlier studies have demonstrated that administration of PPAR-γ ligands such as thiazolidinediones exert insulin sensitizing and anti-inflammatory effects through action on adipocytes and are thus widely used to treat metabolic-related disorders such as T2DM and atherosclerosis among others25. The present results in addition showed a significant increase in hypothalamic and adipose HDAC in obese animals when compared to the control group, which seems to be in agreement with earlier studies that identified many epigenetic regulators, including HDAC as an interacting partner with PPAR-γ66. Similarly, the unliganded PPAR-γ heterodimeric complex is reported to be associated with a multicomponent corepressor complex that contains HDAC activity. After binding to the PPAR-γ ligand, the corepressor complex is dismissed, and the coactivator complex that possesses histone acetylase activity is recruited to the PPAR-γ heterodimer. This leads to chromatin remodeling, which facilitates active transcription67. Therefore, HDAC inhibitor could be a potential agonist of PPAR-γ and a possible suitable therapeutic intervention to obesity.Notably, there was a significant reduction in the plasma, hypothalamic and adipose concentration of PPARS-γ in obese animals compare to the control group. This reduction in PPAR-γ corresponded with increased visceral adiposity and bodyweight as well as impaired hypothalamic and adipose gluco-lipid metabolism, excessive lipid peroxidation, G6PD/GSH and NO depletion, elevated TNF-α/IL-6 and plasma or adipose leptin concentration that characterized obese animals. Similar to the present observations, deficiency of PPARs has been demonstrated in metabolic stress with consequent impaired adipogenic remodeling69, and acetate has been earlier shown to improve insulin sensitivity and exert anti-inflammatory effects against neuroinflammation, hypertension and hyperandrogenism among others70. Similarly, the present results are also in line with the observation of Lee et al., who revealed that the inhibition of HDAC by MPT0E014 attenuated cardiomyopathy by upregulation of PPAR-γ71. However, to the best of our knowledge, the results of the present study suggest for the first time that inhibition of HDAC by acetate ameliorates disrupted hypothalamic-adipose metabolic network with consequent normalization of visceral adiposity in HFD-induced obesity by modulation of PPAR-γ. Although the present study is not without a limitation in such that the molecular mechanism underlying the regulatory role of acetate was not fully investigated. Nevertheless, the present data provides justification for future investigation of molecular mechanism and possibly holds public health interest or clinical relevance for prevention and management of obesity and its comorbidities.Intriguingly, administration of acetate, an inhibitor of HDAC significantly increased plasma, hypothalamic and adipose PPAR-γ concentration with corresponding improvement in insulin sensitivity and glucose regulation as well as hypothalamic and adipose lipid metabolism, lipid peroxidation, G6PD/GSH-dependent antioxidant capacity, NO, inflammatory mediators and normalized leptin concentration, visceral adiposity and body weight in OBS + ACT group compared to untreated obese group. A number of studies have documented acetate as HDAC inhibitorIn conclusion, the present results demonstrate that obesity is characterized with aberrant brain-adipose metabolic network, which is accompanied by altered HDAC and PPAR-γ. The results in addition suggest that acetate, an HDAC inhibitor rescues defective brain-adipose metabolic network with consequent normalization of body weight/visceral fat mass and metabolic homeostasis by modulation of PPAR-γ and suppression of oxidative stress. Hence, nutritional modifications that incorporate acetate engendered high fiber diet might be beneficial to obese individuals.The study was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the protocol was approved by the Ethical Review Board of Afe Babalola University, Ado-Ekiti, Nigeria (ABUADERC/2020/06), and every effort was made to minimize both the number of animals used and their suffering. All the experiments were also performed and reported in accordance with the ARRIVE guidelines 2.0. Ten-week-old male Wistar rats weighing 160–180 g were procured from the animal house of the College of Health Sciences, Afe Babalola University, Ado-Ekiti. Rats had unhindered access to standard rat chow and tap water. After two weeks of acclimatization, the animals were randomly assigned into four groups (n = 6 per group) namely: Control (CTL), ACT-treated group, OBS group and OBS + ACT group. Rats were maintained in a colony under standard environmental conditions of temperature (22–26 °C), relative humidity (50–60%), and 12-h dark/light cycle.72 and administered for 12 weeks.Obesity was experimentally induced by exposing the animals to 40% high fat diet prepared as described previouslypo), ACT-treated group received standard rat chow and sodium acetate , OBS group receive 40% HFD and distilled water . Blood was obtained from the tail before glucose load and then sequentially at 30, 60, 90 and 120 min. Glycemic levels were determined with a hand-held glucometer . Insulin sensitivity was determined using quantitative insulin sensitivity check index (QUICKI), which is expressed as 1/[log (fasting insulin) + log (fasting glucose)74, while IR was determined using its surrogate, triglyceride-glucose index, which is expressed as TyG = Ln (TG (mg/dl) × FPG (mg/dl)/2)75.The OGTT was performed 48 h prior to the sacrifice of the rats. The rats had 12-h overnight fast. Animals were loaded with glucose . Blood was collected by cardiac puncture into heparinized tube and was centrifuged at 704 g for 5 min at room temperature. Plasma was stored frozen until it is required for biochemical assay.At the end of the treatment, the rats were anesthetized with sodium pentobarbital . This method is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule.The plasma and tissue concentration of leptin was determined using Rat ELISA kits with Catalog No. E-EL-R0582 obtained from Elabscience Biotechnology Inc. . Similarly, plasma concentration of ghrelin was determined using Rat ELISA kits with Catalog No. E-EL-R0842 obtained from Elabscience Biotechnology Inc. .76.Triglycerides, TC and HDLc were estimated in the plasma, adipose and hypothalamus by standardized colorimetric methods using kits with Catalog No. BXC0271, BXC0261 and BXC0422A respectively obtained from Fortress Diagnostics Ltd. . Atherogenic indices were estimated as TG/HDLc and TC/HDLc in line with earlier descriptionMalondialdehyde is a marker of lipid peroxidation and GSH reflect cellular antioxidant capacity of the cells. Malondialdehyde was determined from the plasma, adipose tissue and hypothalamus by standard non-enzymatic spectrophotometric method using assay kits from Randox Laboratory Ltd. . This method involves the reaction of MDA in the sample with thiobarbituric acid (TBA) to generate MDA-TBA adduct, which was quantified spectrophotometrically, whereas GSH was determined using a non-enzymatic spectrophotometric method with assay kits obtained from Oxford Biomedical Research Inc. . Glutathione determination by spectrophotometric method was based on the oxidation of GSH in the sample by the sulfhydryl reagent 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5′-thio-2-nitrobenzoic acid (TNB), measured at 412 nm.Glucose-6-phosphate dehydrogenase activity was determined from the plasma, adipose tissue and hypothalamus using standard spectrophotometric method, whereas PDK4 level was determined from the plasma, visceral fat and hypothalamus using Rat ELISA kits obtained from Calbiotech Inc. .Plasma and tissue NO were assayed spectrophotometrically by measuring the accumulation of its stable degradation products, nitrate and nitrite using kits from Oxford Biomedical Research Inc., . This kit employs the NADH-dependent enzyme nitrate reductase for conversion of nitrate to nitrite prior to the quantification of nitrite using Griess reagent—thus providing for accurate determination of total NO production.Plasma and tissue concentration of TNF- α and IL-6 were determined by the quantitative standard sandwich rat ELISA technique using monoclonal antibody specific for these parameters using assay kits with Catalog No. E-EL-R0019 and Catalog No: E-EL-R0015 respectively, obtained from Elabscience Biotechnology Inc. .The adipose and hypothalamic tissue levels of HDAC were determined using Rat ELISA kits with Catalog No. E0791Ra, obtained from Bioassay Technology Laboratory , in compliance with the manufacturer’s procedure.γ was determined by the quantitative standard sandwich ELISA technique using monoclonal antibody specific for this parameter with rat assay kit  obtained from Elabscience Biotechnology Inc. .Plasma and tissue concentration of PPAR- Plasma and tissue concentration of pyruvate and lactate were determined by non-enzymatic standard colorimetric method using assay reagent kits obtained from Randox Laboratory Ltd.  respectively. Whereas, plasma, adipose and hypothalamic GGT activity was measured by standardized enzymatic colorimetric method using assay kit obtained from Randox Laboratory Ltd. .p < 0.05.The data were confirmed to be normally distributed using Shapiro–Wilk test and. All data were represented as means ± SD. Graphpad prism 9.2.0 was used to perform the statistical group analysis, and the mean values were compared using one-way ANOVA except for the oral glucose tolerance, where data were analyzed by repeated measures ANOVA. Then post hoc analysis was performed by Bonferroni’s test and statistically significant difference was determined at The study was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the protocol was approved by the Ethical Review Board of Afe Babalola University, Ado-Ekiti, Nigeria (ABUADERC/2020/06), and every effort was made to minimize both the number of animals used and their suffering.Supplementary Information.


Global Deterioration Scale scores were measured 1 month post incident and used to place the patients in either favorable or poor outcome groups. Initial serum GDF15 and 8-OHdG concentrations were significantly increased in the poor-outcome group and all four biomarkers decreased at 24 h post HBO2 therapy, and were then maintained or further decreased at the 1-week mark. Notably, the degree of change in these biomarkers between baseline and 24 h post HBO2 were significantly larger in the poor-outcome group, reflecting greater CO-associated stress, confirming that post-CO poisoning serum biomarker levels and their response to HBO2 were proportional to the initial stress. We suggest that these biomarkers accurately reflect neuronal toxicity in response to CO poisoning, which is consistent with their activity in other pathologies.Mitochondrial and oxidative stress play critical roles in the pathogenic mechanisms of carbon monoxide (CO)-induced toxicity. This study was designed to evaluate whether the serum levels of specific stress biomarkers might reflect brain injury and act as prognostic markers for the development of neurocognitive sequelae following CO poisoning. We analyzed the data from 51 adult patients admitted with acute CO poisoning and measured the serum level expression of growth differentiation factor 15 (GDF15) and fibroblast growth factor 21 (FGF21), indicators of mitochondrial stress, and 8-Oxo-2′-deoxyguanosine (8-OHdG) and malondialdehyde (MDA), indicators of oxidative stress. Serum was collected upon arrival at the hospital, at 24 h post treatment, and within 7 days of HBO A . A 47]. 2 therapy as primary indicators in this study, comparing these values to assess whether HBO2 reduced stress. The secondary outcome of this study was the evaluation of these biomarkers as prognostic tools for neurocognitive outcomes 1 month post trauma.We used serum mitochondrial (GDF15 and FGF21) and oxidative stress (8-OHdG and MDA) biomarker levels pre- and post-HBOt-test or Mann–Whitney U test for continuous variables and the chi-square or Fisher’s exact tests for categorical variables. We used pairwise repeated-measure analysis of variance to assess individual changes in serum biomarker levels between the baseline and follow-up samples. These evaluations were completed using a Friedman test and Dunn’s multiple comparison correction. Variables with a p < 0.1 in the univariate analyses were then evaluated in the multivariate analysis using stepwise selection. The prognostic powers of prediction models were evaluated using area under the receiver operator characteristic curve (AUROC) analyses, with assessments of likelihood ratios, predictive values, and accuracy. The optimal cut-off values were determined using the Youden index and all statistical analyses were performed using SPSS 25.0 software  and Prism 8.0 . Two-sided p values < 0.05 were considered statistically significant.Data for continuous variables were presented as the median (interquartile range) or mean ± standard deviation while categorical variables were recorded as frequencies (percentages). Continuous data were tested for normality using the Shapiro–Wilk test and subgroups were compared using independent 2 therapy are proportional to the initial stress imposed by CO poisoning and we suggest that serial measurements of these biomarkers could provide more information around the pathological stress burdens and disease progression of neurocognitive disorders and facilitate improved prognostic evaluations in not only acute CO toxicity but also other chronic metabolic and neurodegenerative diseases.This study suggests that serum biomarkers for mitochondrial and oxidative stress correlate with the neurocognitive outcomes of CO poisoning. The changes in these biomarkers following HBO


Genomic profiling of cancer-derived materials in circulation has become an alternative approach for tumour genotyping. The detection of tumour origin markers such as DNA methylation in bodily fluids enables cancer screening, early-stage diagnosis and evaluation of therapy response. The development of broad platform technologies that underpin many in vitro clinical diagnostic tests has brought about a paradigm shift in cancer management and diagnosis. This study developed a multifaceted technology platform based on bioengineered polymer nanobeads for efficient capture and electrochemical detection of DNA methylation in ovarian cancer patient samples. This could be a versatile diagnostic platform for detecting numerous disease biomarkers, thus allowing several disease diagnoses.2O2/HRP/HQ) redox cycling system. In the presence of methylated DNA, the enzymatically produced (in situ) metabolites, i.e., benzoquinone (BQ), binds irreversibly to cellular DNA resulting in the unstable formation of DNA adducts and induced oxidative DNA strand breakage. These events reduce the available BQ in the system to support the redox cycling process and sequel DNA saturation on the platform, subsequently causing high Coulombic repulsion between BQ and negatively charged nucleotide strands. Thus, the increase in methylation levels on the electrode surface is inversely proportional to the current response. The method could successfully detect as low as 5% methylation level. In addition, the assay showed good reproducibility (% RSD ≤ 5%) and specificity by analysing various levels of methylation in cell lines and plasma DNA samples from patients with ovarian cancer. We envision that our bioengineered polymer nanobeads with high surface modification versatility could be a useful alternative platform for the electrochemical detection of varying molecular biomarkers.DNA methylation is a cell-type-specific epigenetic marker that is essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. It is also responsible for the pathogenesis of many diseases, including cancers. Herein, we present a simple approach for quantifying global DNA methylation in ovarian cancer patient plasma samples based on a new class of biopolymer nanobeads. Our approach utilises the immune capture of target DNA and electrochemical quantification of global DNA methylation level within the targets in a three-step strategy that involves (i) initial preparation of target single-stranded DNA (ss-DNA) from the plasma of the patients’ samples, (ii) direct adsorption of polymer nanobeads on the surface of a bare screen-printed gold electrode (SPE-Au) followed by the immobilisation of 5-methylcytosine (5mC)-horseradish peroxidase (HRP) antibody, and (iii) immune capture of target ss-DNA onto the electrode-bound PHB/5mC-HRP antibody conjugates and their subsequent qualification using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H DNA methylation is essential for several cellular regulatory pathways such as X-chromosome inactivation, genomic imprinting, long term gene silencing and regulation of chromatin structure . All the electrodes were grounded with carbon tape to prevent charging. Charge compensation was also used to record the wide and high-resolution data from each sample6-PHB nanobeads as previously described [g for four minutes between each wash in order to remove the ethanol. All samples were re-suspended in 500 μL 1× PBS buffer, and 5 mg of human IgG  was added to each sample and incubated at 25 °C for 30 min with agitation to allow IgG binding to occur. After incubation, the tubes were centrifuged at 6000× g for four minutes and the unbound fraction of IgG in supernatants were aliquoted and analysed. The sediment was washed three times with 1× PBS buffer by pipetting, subsequently followed by centrifugation at 6000× g for four minutes between each wash. Bound IgG from PHB nanobeads was eluted by re-suspending the sediment in 0.5 mL of 50 mM Glycine (pH 2.7) and incubated at room temperature for five minutes with agitation. Then, the samples were centrifuged at 16,200× g for four minutes, and the eluted IgG in the supernatant was neutralised by adding 10 μL of 1 M K2HPO4 and analysed. Protein concentrations were analysed using the Bradford assay.An IgG-binding capacity assay was used to assess the functionality of Zescribed . PHB nan2O2/HRP/HQ redox cycling system was then used to achieve an electrochemical quantification of methylated DNA sequences present in the cell line and plasma solutions. The functionality of the assay was determined by comparing the assay performance in detecting 100% of methylated DNA target sample. As observed in −2) respectively. The increased current response for WGA could be due to the HRP saturation level on the electrode surface, which allows a huge amount of available HQ to participate in the redox cycling processes. In the presence of methylated DNA (100%), a significant decrease in current response is observed. This may be attributed to the quinone metabolite generated in the redox cycling process, which facilitated the irreversible formation of unstable DNA adducts resulting in the deficiency of available metabolite in the redox cycling system. This may, therefore, steer a decrease in the current response. In addition, the oxidative DNA strand breaks induced by the electrophilic attack on DNA by BQ saturate the electrode surface with DNA, thereby increasing the Coulombic repulsion between BQ and negatively charged DNA [rged DNA ,37. The To confirm the direct adsorption of PHB nanobeads onto a bare SPE-Au surface, AFM of the electrode was recorded. The nominal height of PHB beads is depicted in To further characterise the surface of the electrode after each modification step, XPS was recorded. The initial high-resolution spectrum of cleaned bare SPE-Au electrode indicates the presence of carbon on its surface a; hence 2) of 0.9917. A methylation change as low as 5% could be detected. This data can be accredited to PHB nanobeads’ superior physicochemical properties that provide a huge active surface area for antibody and target immobilisation. The relative standard deviation of three independently fabricated sensors for detecting 10 ng uL−1 methylated target DNA was <5%, indicating acceptable precision and fabrication reproducibility. Comparable results have also been reported previously based on colourimetry [Global DNA methylation plays a major role in the pathogenesis of various types of genes by altering their expression ,45,46. Aurimetry ,50 and gurimetry ,51 affinurimetry . In thisurimetry  and massurimetry . However−2) compared to WGA (32.7 µA cm−2), indicating that DNA sequences derived from SKOV3 and OVCAR3 could be hypermethylated at the promoter gene. The chronoamperometric analysis shows that the current density changes derived from the cell lines are easily detectable against that of the control. (−2) is much lower than that of SKOV3 and OVCAR3, indicating hypomethylation at the promoter gene. These results demonstrate that SKOV3 exhibits over 35% methylation and OVCAR3 more than 41%. The cell line data shows good reproducibility of our assay (% RSD of <4.25% for n = 3) for the inter-assay signals for analysing DNA methylation levels in the ovarian cancer cell line without prior amplification or pre-treatment. The methylation statuses obtained for the cell lines indicate that the proposed assay may be an alternative for detecting global methylation in cell-derived samples.Metastasis, the process of cancer dissemination, is responsible for at least 90% of cancer deaths. Treating or preventing metastasis remains a long-standing aim in cancer research. However, tumours are a complex mixture of cancer cells which hinders the success of therapy. To understand what factors contribute to metastasis and treatment response, it is critical to characterise the tumour microenvironment (non-cancerous cells in the tumour tissue). Ovarian cancer offers a unique opportunity to study the tumour microenvironment because diagnosis often occurs at advanced stages when metastasis has already taken place. To test the applicability of our assay for detecting methylation levels in complex biological samples, DNA samples derived from two ovarian cancer cell lines (SKOV 3 and OVCAR 3) and one non-cancerous cell line (MeT-5A) were tested . A fullycontrol. b. This rThe applicability of our assay to analyse clinical samples was demonstrated by analysing six plasma DNA samples (P1–P6) derived from patients with epithelial ovarian cancer. Two ovarian benign tissue DNA samples (B1 and B2) were also used as a control. As depicted in This study describes the development of a simple and new method for quantifying DNA methylation events using PHB nanobeads. The method illustrates the application of PHB nanobeads as electrode surface modifiers, which facilitate the adsorption of a specific antibody for methylated DNA. The detection was achieved by direct immobilisation of various DNA samples onto a 5mC/HRP-PHB nanobead modified electrode. This method eliminates several modifications and functionalisation steps involved in conventional assays. In addition, it also avoids PCR amplification and the need for sequencing analysis. Most importantly, we have demonstrated the clinical feasibility of our method to detect methylation levels in ovarian cell lines and clinical samples from ovarian cancer patients. Unlike conventional methods, our assay’s high analytical efficiency, ease of use and low sample input may be useful for routine clinical diagnostics and a variety of other applications. It is worth emphasising that our assay design was to measure the samples’ total DNA methylation content  and not methylation of the CpG island of a specific gene. However, the assay can be modified to target gene-specific methylation.Considering the great importance of DNA methylation in biological processes, our assay may present an important step towards developing a biosensor that could be useful for detecting disease-specific methylated DNA in bodily fluids . Given that PHB nanobeads have high surface modification versatility for efficient capture and detection of different targets, we believe that the proposed assay could also be a multifaceted diagnostic platform for detecting a myriad of disease biomarkers, thus enabling multiple diseases diagnoses.


Myeloid diseases are often characterized by a disturbed regulation of myeloid cell proliferation, survival, and maturation. This may either result in a severe paucity of functional neutrophils (neutropenia), an excess production of mature cells (myeloproliferative disorders) or in clonal expansions of dysplastic or immature myeloid cells (myelodysplasia and acute myeloid leukemia). Although these conditions can be regarded as separate entities, caused by the accumulation of distinct sets of somatic gene mutations, it becomes increasingly clear that they may also evolve as the prime consequence of a congenital defect resulting in severe neutropenia. Prominent examples of such conditions include the genetically heterogeneous forms of severe congenital neutropenia (SCN) and Shwachman-Diamond Syndrome. CSF3 treatment is a successful therapy to alleviate neutropenia in the majority of these patients but does not cure the disease nor does it prevent malignant transformation. Allogeneic stem cell transplantation is currently the only therapeutic option to cure SCN, but is relatively cumbersome, e.g., hampered by treatment-related mortality and donor availability. Hence, there is a need for new therapeutic approaches.Developments in disease modeling, amongst others based on induced pluripotent stem cell and CRISPR/Cas9 based gene-editing technologies, have created new insights in disease biology and possibilities for treatment. In addition, they are fueling expectations for advanced disease monitoring to prevent malignant transformation.This review highlights the recent progress made in SCN disease modeling and discusses the challenges that are still ahead of us to gain a better understanding of the biological heterogeneity of the disease and its consequences for patient care. Hereditary bone marrow (BM) failure syndromes prone to the development of myeloid malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) have been the subject of study for many decades. The introduction of advanced genetics based on next generation sequencing technologies has advanced our understanding of the biology of these conditions and has improved diagnosis, prognostication and disease monitoring ,2. Likew5/L . Importantly, mutant ELANE was highly expressed in these cells, which was not seen in HAX1-SCN or in ELANE-SCN without predicted misfolding mutations. Intriguingly, deletion of PML by CRISPR/Cas9 resulted in the loss of ELANE expression and restored the response of the HPCs to CSF3 in colony forming assays. Taken together, these results suggest that the lack of neutrophil recovery in response to CSF3 therapy in certain ELANE-SCN cases, particularly those with misfolding mutations, may be attributable to elevated PML expression as a result of excessive oxidative stress in the HSPC compartment [▪▪].As an alternative pathway for the classical UPR, the promyelocytic leukemia protein (PML) has been reported to interact with and degrade misfolded proteins. This particularly occurs under conditions of excessive oxidative stress and elevated production of reactive oxygen species, leading to the generation of PML nuclear bodies (PML-NBs) . A recenpatients ▪▪. Imporpartment ▪▪.AK2, showed that the mutation caused excessive oxidative stress in HSPCs. This could be reversed by antioxidant treatment, which restored differentiation of the AK2-deficient iPSCs into mature granulocytes . It was elegantly shown in these papers that the loss of EIF6 protein compensates the ribosome assembly defect caused by the loss of the Shwachmann-Bodian-Diamond Syndrome protein and restores ineffective hematopoiesis by increasing fitness of the SDS deficient HSPCs. In contrast to mutations in p53, which arise in SDS HPSC and contribute to leukemic progression .In iPSC models of SDS, hyperactive TGFβ signaling contributes to defective myeloid development, which can partly be restored by adding a TGFβ receptor small molecule inhibitor . Anotherpatients ▪▪,54▪▪. gression , SGR by ELANE allele accepting that the wild-type allele will also be deleted, resulting in complete loss of NE expression [56▪▪]. This is considered acceptable given that patients suffering from Papillon-Lefevre syndrome, who lack functional NE and other neutrophil serine proteases, present with relatively mild symptoms . Notably, off-target mutations were undetectable, indicating that the procedure may eventually qualify for safe application in patients [▪▪].Conceivably the most promising therapeutic options to restore normal hematopoiesis in congenital neutropenia involve CRISPR-Cas9 based gene correction of HSPCs, combined with autologous stem cell reinfusion. This is particularly challenging in ELANE-SCN, given the wide variety of mutations. To cope with this, one strategy aims at deleting the mutant pression ,56▪▪. Thsymptoms . A promiutations ▪▪. Notabpatients ▪▪.Although altogether fueling expectations, it is reasonable to state that the advances in therapeutic options to alleviate neutropenia based on disease modeling and genome editing are still relatively modest and await systematic and collaborative efforts from clinicians/pediatricians and clinical-translationally oriented researchers to further develop and implement these approaches into clinical protocols. Until this has been achieved, CSF3 therapy  remains the prime treatment to avoid life-threatening infections in SCN.CSF3R are frequently observed in ELANE-SCN and HAX1-SCN patients who progress to MDS or AML. These mutations most often cause an intracellular truncation of the CSF3R protein leading to significant alterations in signaling behavior and can arise in minority clones years before leukemic progression occurs [RUNX1 in clones already harboring CSF3R mutations [Mutations in n occurs ,51. Onceutations .CSF3R and RUNX1 mutations and sustained CSF3 treatment on leukemic progression [CSF3R and RUNX1 mutations in their HSPCs exactly as those identified in a case of SCN/AML displayed an excess of immature myeloblasts (>10%) in the peripheral blood after 3 weeks of CSF3 treatment, which was not seen in the absence of either of the mutations or without CSF3 treatment [CXXC4 and reduced expression of TET2 protein. Comparative RNAseq analysis of the mouse AML and the patient SCN/AML cells revealed highly similar transcriptional profiles indicative of the activation of multiple inflammatory pathways. This and a related study [CSF3R and RUNX1 mutations, which is then stepwise aggravated and involving additional inflammatory pathways  upon acquisition of the mutations associated with leukemic progression. These combined observations in mouse and patient models support a link between inflammation to leukemia predisposition and clonal evolution toward MDS/AML in SCN  [BAALC expression and/or MKa2 phosphorylation may prevent transformation or eliminate AML blasts in SCN/AML [Finally, iPSC modeling of specific phases of leukemia progression has identified upregulation of a (MK2a) ▪. Hence, SCN/AML ▪.CSF3R, RUNX1, ASXL1) and cytogenetic abnormalities  act in concert with elevated inflammation in HSPCs may open avenues for new anti-inflammatory therapies to prevent malignant progression or to eradicate SCN/AML blasts, similar to what has been proposed for MDS and MPN. Likely, the highest expectations for curative therapies should come from the recent developments in gene editing technologies, as for instance currently being clinically explored in sickle cell disease [This review has summarized the recent developments in disease models and gene editing technologies that contributed to the progress made in understanding the biology of SCN and its predisposition to leukemia. Yet, many issues are still unresolved. First, it remains unknown to what extent cell extrinsic (‘niche’) factors contribute to disease development, as has for instance been implicated in SDS, MDS, and MPN ,66–69. S disease .I acknowledge members of the Department of Hematology at Erasmus MC and the EuNet-INNOCHRON network for discussions.No financial support was required for this review article.There are no conflicts of interest.


Scarce drug penetration in solid tumours is one of the possible causes of the limited efficacy of chemotherapy and is related to the altered tumour microenvironment. The abnormal tumour extracellular matrix (ECM) together with abnormal blood and lymphatic vessels, reactive stroma and inflammation all affect the uptake, distribution and efficacy of anticancer drugs.We investigated the effect of PEGylated recombinant human hyaluronidase PH20 (PEGPH20) pre-treatment in degrading hyaluronan , one of the main components of the ECM, to improve the delivery of antitumor drugs and increase their therapeutic efficacy. The antitumor activity of paclitaxel (PTX) in HA synthase 3-overexpressing and wild-type SKOV3 ovarian cancer model and in the BxPC3 pancreas xenograft tumour model, was evaluated by monitoring tumour growth with or without PEGPH20 pre-treatment. Pharmacokinetics and tumour penetration of PTX were assessed by HPLC and mass spectrometry imaging analysis in the same tumour models. Tumour tissue architecture and HA deposition were analysed by histochemistry.Pre-treatment with PEGPH20 modified tumour tissue architecture and improved the antitumor activity of paclitaxel in the SKOV3/HAS3 tumour model, favouring its accumulation and more homogeneous intra-tumour distribution, as assessed by quantitative and qualitative analysis. PEGPH20 also reduced HA content influencing, though less markedly, PTX distribution and antitumor activity in the BxPC3 tumour model.Remodelling the stroma of HA-rich tumours by depletion of HA with PEGPH20 pre-treatment, is a potentially successful strategy to improve the intra-tumour distribution of anticancer drugs, increasing their therapeutic efficacy, without increasing toxicity.The online version contains supplementary material available at 10.1186/s13046-021-02070-x. Many molecular mechanisms responsible for resistance to anticancer drugs have been elucidated over the years. The co-existence of heterogeneous populations of cancer cells with different sensitivity explains why, even after an initial response, most solid tumours relapse, becoming resistant to treatments.Growing evidence suggests that the resistance of many solid tumours can also be due to insufficient and heterogeneous tumour drug distribution , 2. StudThe penetration capacity of a drug depends on its physical-chemical properties and on the neoplastic cells’ characteristics, but the low delivery in tumour tissue is mainly related to the altered tumour microenvironment (TME) . AbnormaHA is a glycosaminoglycan and a major component of the normal ECM, whose accumulation is significantly increased in several solid malignancies, particularly in advanced stage disease . High HATo improve the delivery of antitumor drugs and increase their therapeutic efficacy, some strategies based on ECM modification have been tested. Lowering HA levels in TME with a hyaluronidase has been suggested as a “distribution enhancing approach”. By cleaving polymeric HA into substituent units, hyaluronidase can liberate trapped water molecules, thus rapidly lowering TIFP and enabling collapsed vessels in the tumour to open. Thus, it would improve tumour perfusion and drug permeation. rHuPH20, a recombinant human hyaluronidase approved for subcutaneous injection to facilitate the absorption of other injected drugs  or fluids, has been further developed for anticancer purposes. PEGPH20 is an investigational PEGylated form of rHuPH20 that has a longer plasma half-life (about 10.3 h for PEGPH20 compared to 3 min for rHuPH20 in mice) and it is suitable for intravenous administration . In precIn patients, PEGPH20 is tolerated, although prophylactic steroids and anticoagulant are required to prevent musculoskeletal and thromboembolic events , 32. TumA fuller description of the connections between drug distribution, TME characteristics and antitumor activity could help elucidate the true potential of this therapeutic approach. We investigated the distribution of paclitaxel (PTX) after PEGPH20 pre-treatment using mass spectrometry imaging (MSI) and correlated it with antitumor activity and tissue modifications in different preclinical models.Paclitaxel  and paclitaxel-D5  were dissolved in ethanol at a concentration of 1 mg/ml. Serial dilutions of drug were prepared in 50% ethanol from 0.5 to 100 pmol/μl for all MSI experiments.For treatment purposes, PTX was dissolved in 50% Cremophor EL (Sigma) and 50% ethanol and further diluted 1:5 in saline immediately before use.The PEGPH20 formulation  consisted of the active compound dissolved in histidine buffer  at 5 mg/ml. It was diluted in saline solution (NaCl 154 mM) just before the treatment.2 nanoparticles  were used as a matrix for MSI experiments, dissolved at the concentration of 1 mg/ml in ethanol 50%/KCl 0.5%. The TiO2 nanoparticle suspension was vortexed and sonicated for 3 min just before use, to reduce agglomeration and sedimentation.TiOParental SKOV3 (SKOV3) and HAS3-overexpressing SKOV3 (SKOV3/HAS3) cell lines were provided by Halozyme Therapeutics Inc. SKOV3/HAS3 were obtained by transducing SKOV3 cells with a retrovirus carrying human HA synthase 3 (HAS3) cDNA. These cells are characterized by production of high HA levels. Both cell lines were maintained in McCoy medium added with 10% FBS and glutamine. The BxPC3 pancreatic adenocarcinoma cell line was maintained in RPMI with 10% FBS and glutamine. All the cell lines were mycoplasma free and authenticated by short-tandem repeat (STR) profiling.-nu/nu mice were obtained from Envigo RMS. They were maintained under specific pathogen-free conditions, housed in isolated vented cages, and handled using aseptic procedures after an acclimatization period of one week.Six- to 7-week-old female NCrARRIVE guidelines 2.0 .All procedures involving animals and their care were conducted in conformity with the following laws, regulations, and policies governing the care and use of laboratory animals: Italian Governing Law ; Mario Negri Institutional Regulations and Policies providing internal authorization for persons conducting animal experiments ; the NIH Guide for the Care and Use of Laboratory Animals (2011 edition) and EU directives and guidelines (EEC Council Directive 2010/63/UE) and in line with Guidelines for the welfare and use of animals in cancer research {Workman, 2010}. Animal experiments were reviewed and approved by the IRFMN Animal Care and Use Committee (IACUC) that included “ad hoc members for ethical issues. Animals were housed in the Institute’s Animal Care Facilities, which meet international standards; they are regularly checked by a certified veterinarian who is responsible for health monitoring, animal welfare supervision, and experimental protocols and procedures revision. Experiments were reported following the 6 cells) were implanted subcutaneously in the flank of nude mice. Tumour growth was measured with a Vernier caliper two/three times a week, and tumour weights (1mm3 = 1 mg) were calculated as [length (mm) * width2 (mm2)]/2. When tumours reached approximately 150–200 mg, mice were assigned to the experimental group by stratified randomization based on tumour weight (8 to 9 mice/group) and treated with PEGPH20  alone or with PTX  or with PEGPH20 followed by PTX . Control groups received the same volume of the respective vehicles. Mice were weighted two/three times a week to evaluate general toxicity. Tumour volume was measured thrice a week up to the ethical endpoint (1500 mm3) then mice were euthanized. For the analysis of the tumour growth curves, each tumour weight (TW) measure was normalized to the tumour weight of the same mouse at the start of treatment . Antitumor efficacy was expressed as best T/C% were T and C were the mean of the RTWs of treated and control mice, respectively according to the standards of the National Cancer Institute (NCI) of the United States  were calculated as follows: k = [log TWt(i + 1)-log TWt]/ (t(i + 1)-ti). The experimental groups were compared two by two using a Wilcoxon Rank-Sum test, stratified by intervals, on the obtained k values . Student’s t-test for unpaired samples was used to evaluate differences in drug distribution experiments. Differences in the pharmacokinetic profiles were analysed by two-way ANOVA (Graph Pad Prism 8).The treatment effect on xenograft tumour growth curves was formally tested using a non-parametric approach. For each mouse, the partial tumour growth rates (k) in every interval [tp-value< 0.001 compared to control, p-value< 0.001 compared to single treatments). An in depth analysis of the tumour growth curves is shown in Additional file The effect of PEGPH20 pre-treatment on PTX antitumor activity was assessed in parental and HAS3-overexpressing SKOV3 tumours Fig. . PTX indTo verify the hypothesis that PEGPH20 enhances the PTX antitumor efficacy by improving its distribution in tumour tissue, we performed an in-depth analysis of spatial distribution of PTX within tumour tissue, using an in-house developed MSI technique. Measures of drug concentrations in tumour homogenates and plasma pharmacokinetics are not fully indicative of drug distribution in tumour tissue. Since heterogeneous penetration of pharmacological compounds in neoplastic tissue is a known mechanism of drug resistance, the determination of drug intratumor penetration adds valuable information. The MSI analysis of PTX is shown in Fig. MSI images were further processed to extract quantitative parameters describing drug distribution. In addition to the mean and CV% of the drug concentration calculated at pixel level, we examined a panel of GLSZM features selected for their significance in relation to the diffusion and homogeneity of the drug in the tissue. The complete panel of GLSZM features and an interpretation of their meaning in this context is reported in Additional file The complete pharmacokinetic analysis after a single dose of PTX 20 mg/kg, alone or after PEGPH20 pre-treatment in both tumour models is reported in the Additional Information. PEGPH20 pre-treatment minimally affect PTX pharmacokinetic behaviour. Indeed, PTX concentrations in tumour samples collected at different time points were comparable in the two different experimental groups (with or without PEGPH20 pre-treatment). Similar levels were also found for plasma in the SKOV3 model while circulating PTX was lower after PEGPH20 than vehicle pre-treatment in SKOV3/HAS3 model but only 1 h after PTX. Similarly, PTX levels in liver were lower in the combination in both experimental models 1 h after PTX.To determine whether the different PTX distribution in tumour was due to differences in tissue architecture, we did histological examinations on tumour sections adjacent to those analysed by MSI. H&E staining confirmed that SKOV3 tumours were composed of sheets/solid areas of neoplastic cells arranged in lobules, infiltrated by a moderate to large amount of thin fibrovascular stroma and occasionally thicker fibrous septa Fig. . VariablIn SKOV3/HAS3 tumours, a looser arrangement of the tissue structure was evident, with loss of defined lobules. HA ability to bind large amounts of water molecules might be responsible for the looser arrangement of the tumour tissue observed in SKOV/HAS3 samples: a large amount of water is frozen and released during sample preparation, producing the artefacts characterized by clear/empty spaces dissecting the tumour tissue. The PEGPH20 pre-treatment restored a more compact tumour architecture, similar to that of parental SKOV3 tumours Fig. . No diffp-value = 0.025).Specific HA depletion in SKOV3/HAS3 tumours following PEGPH20 treatment was quantified in tissue sections stained for HA using the immuno-adhesin HTI-601 with DAB used as the chromogen Fig. . A high MSI analysis was done following the same therapeutic regimen as PEGPH20 and PTX used for antitumor activity experiments . The results confirmed that PEGPH20 pre-treatment improved drug distribution in SKOV3/HAS3 but there were no differences between the two treatments in the parental SKOV3 model Fig. . The comThe clinical relevance of our results was verified by testing the effect of PEGPH20 on PTX distribution in a xenograft model of pancreatic cancer, a tumour known for its dense, desmoplastic stroma with abundant ECM. The MSI analysis of PTX results are shown in Fig. Quantitative analysis of the MSI images indicated that PEGPH20 in BxPC3 induces a pattern of modifications in our panel of features similar to that of the SKOV3/HAS3 model: an increase of the mean pixel drug concentration, with lower LZE, higher HGZE and lower IV Fig. . These ap-value 0.019; Fig. No difference was seen between the experimental groups in PTX total concentrations measured by HPLC in tumour homogenates Fig. . The BxPHeterogeneous spatial distribution of drugs in solid tumours represents a sort of pseudo-chemoresistance, because tumour cells can survive therapy simply because of lack of exposure . This isHA is a TME component that is often overexpressed in various tumours , 22. HA PEGPH20 was developed for its ability to disrupt HA in tumour tissue, inhibiting HA’s protumor properties, reducing TIFP and solid stress. Indeed, PEGPH20 can be exploited in a combination strategy, to improve the penetration of concomitant drugs through tumour tissue. Previous preclinical studies have shown that PEGPH20 increases the delivery of doxorubicin in pancreatic cancer  and favoIn our study, we selected a HA-overexpressing ovarian cancer cell line and its parental counterpart as tumour models with different HA contents. Cells were implanted ectopically in nude mice. PEGPH20 pre-treatment increased PTX antitumor activity in the ovarian cancer model with high HA levels. In the parental model expressing lower HA levels, PEPGH20 did not affect PTX efficacy. Consistently with clinical data, we also found that the PTX monotherapy was less efficacious in ovarian cancer with high HA levels than in the parental cell line, supporting the correlation between HA content and tumour aggressiveness, chemoresistance and worse prognosis .A single pre-treatment with PEGPH20 did not significantly increase PTX levels in the tumour, although there was a positive tendency. Plasma and liver levels of PTX were also similar, or even lower, in PEGPH20-pretreated mice than control ones. These pharmacokinetic results were really consistent with the reported data on effect of PEGPH20 pre-treatment on systemic and tissue distribution PEGylated polystyrene nanoparticles for drug delivery in HA expressing tumours . InsteadThe effect of PEGPH20 on tumour PTX distribution and antitumor activity was at least partly confirmed in the BxPC3 xenograft, even though the results were less striking. Also in this model, PEGPH20 reduced HA tumour content and slightly improved PTX distribution and antitumor efficacy.The data obtained in the pancreatic model may help to interpret the contradictory results of clinical trials. A phase II trial in patients with HA-high metastatic PDA (HALO 202)  showed sNonetheless, on the basis of our preclinical data, further clinical development of PEGPH20 should not be excluded, as certain tumours (e.g. ovarian cancer) and therapeutic settings could benefit. Further studies are still required to understand the interplay between TME, tumour cells and drug distribution better.This study demonstrated that PEGPH20 pre-treatment can improve PTX distribution in tumour tissue in two models with high HA levels, and ultimately increase PTX antitumor efficacy. Thus remodelling the extracellular matrix of HA-rich tumours is a promising new strategy to improve the intratumor distribution of anticancer drugs, increasing their therapeutic efficacy, without increasing toxicity.Additional file 1. Additional methods and results sections.


Picornaviridae family. The NGS data analysis identified a number of viruses that were missed by the diagnostic panel , and these failures could be attributed to PCR primer/probe binding site mismatches. Unexpected viruses identified included parvovirus B19, enterovirus D68, coxsackievirus A16 and A24 and rubella virus. The regular application of such viral NGS could help evaluate assay performance, identify molecular causes of missed diagnoses and reveal gaps in the respiratory virus set used for local screening assays. The results can provide actionable information to improve the local pneumonia diagnostics and reveal locally important viral pathogens.Pneumonia remains a major cause of mortality and morbidity. Most molecular diagnoses of viruses rely on polymerase chain reaction (PCR) assays that however can fail due to primer mismatch. We investigated the performance of routine virus diagnostics in Kilifi, Kenya, using random-primed viral next generation sequencing  on respiratory samples which tested negative for the common viral respiratory pathogens by a local standard diagnostic panel. Among 95 hospitalised pneumonia patients and 95 household-cohort individuals, analysis of viral NGS identified at least one respiratory-associated virus in 35 (37%) and 23 (24%) samples, respectively. The majority  belonged to the  Polymerase chain reaction (PCR) methods are frequently used for viral diagnosis but costs and logistics limit the number of pathogens included in diagnostic panels. Additionally, mismatches in primers can cause missed diagnoses with evolving RNA pathogens3. Other viruses which are not included in the diagnostic panels due to costs may also contribute to missed diagnoses. Studies in Kilifi  to investigate viral aetiologies of pneumonia in paediatric patients and in the community identified respiratory viruses with these methods9; however, a proportion of samples collected from symptomatic children were negative for any respiratory viruses in the diagnostic screening panel.Pneumonia is a leading cause of illness globally11 without prior knowledge or specific PCR primers. The data generated from agnostic viral metagenomics of clinical samples were used to control the quality of the diagnostics (e.g. determine the presence of unanticipated viruses and variants of unexpected viruses on diagnostic panel), in order to help define the sensitivity of the diagnostic panels, reveal additional common respiratory pathogens, and improve future diagnostic assays for the region. These data will also help infectious disease clinicians to suspect potential causes that are not routinely included in the diagnosis if the clinical presentations warrant such a diagnosis.We evaluated the ability of next-generation sequencing (NGS) to identify viruses missed by the diagnostic panel by evaluating samples from patients with respiratory disease which tested negative for common respiratory viral pathogens. A random priming (agnostic) NGS method was employed directly on clinical samples to detect viruses present in the samples by viral metagenomicsAs shown in Table The number of respiratory viruses (with contigs ≥ 1000 nt) that had been missed by the standard diagnostic panel included 28 human rhinovirus (HRV) in 27 samples , one human parainfluenza virus 1 (HPIV-1) , three RSVB , and six human metapneumovirus (HMPV)  , species B , species C , and species D . The Parvoviridae Human bocavirus (HBoV) and parvovirus B19 (B19) were identified in four and one sample  infection is frequent in young childrenes Table , panel BEnterovirus genus, Picornaviridae family . Apart from HRV (N = 28), viruses from the Enterovirus species were not included in the routine screening.Among 64 viruses detected, the majority were from the Enteroviruses identified were Rhinovirus species A, B and C, with 28 complete or partial genomes identified. All three sets of diagnostic primers used at the time showed multiple mismatches with the genome target sites that could account for the 28 missed HRV cases 14. The 22-month old patient infected with CV-A16 was hospitalized at KCH with pneumonia, but presented no clinical HFMD symptoms, and was discharged home after 3 days. Phylogenetically, the patient’s CV-A16 virus genome was closely related to a CV-A16 strain identified from an Ethiopian child in April 2016 , enterovirus 71 (EV-71) and several additional 16  was identified in six samples, all from a 2-month period (8 April thorugh 3 June 2010) in the household study Fig. . The sixThe detection of human poliovirus type 2 was likely due to viral shedding after oral poliovirus vaccination. The child in whom this isolate was detected was 6 weeks old at the time of sampling, and had received a dose of oral polio vaccine (OPV) 4 days prior. The child was hospitalised with cough and difficulty breathing, and was discharged home after 3 days. The human poliovirus 2 genome obtained was identical to the vaccine strain Sabin 2 (GenBank AY184220). Health authorities were informed about this finding.Four EV-D68 were identified in the hospitalised cohort over a 5-month period (8 April through 318 July 2010). Two of these patients were co-infected with additional Enterovirus strains, CV-A16 or HRV-A. The four children with EV-D68 were hospitalised with severe pneumonia for 3–6 days, and were discharged with no report of neurological symptom.Phylogenetic analysis Fig. , panel CMatonaviridae family, Rubivirus genus, infecting people of any age. However, primary RVi infection during the first trimester of pregnancy may result in congenital rubella syndrome (CRS) or miscarriage. Common sequelae of CRS include deafness, glaucoma and retinopathy and heart defects. Rubella infections can be prevented by highly effective rubella vaccine. In Kenya, national rubella vaccination was not implemented until October 2016 and there was no surveillance of RVi prevalence or CRS incidence17.Rubella is a contagious typically mild disease caused by rubella virus (RVi), a single-stranded RNA virus in the RVi was detected in two KCH patients through this NGS study Table . They weWe identified parvovirus B19 (B19) in a 5-month old hospitalised patient with very severe pneumonia and anaemia. Tests for malaria were negative, and the patient was discharged after 3 days. The identified B19 virus genome belonged to genotype 1A, similar to other global B19 sequences as shown in the phylogenetic tree comparing the local B19 genome to global sequences  type 1 was identified in a hospitalised child with malnutrition, severe pneumonia and diarrhoea, and in three children with upper respiratory infections from different households. These 4 HBoV1 genomes clustered in 2 sub-lineages within genotype 1 when compared with all global sequences as shown in Supplementary Fig. Viruses detected at low frequency included HPIV-1 (one case), human parechovirus (one case), human herpesvirus 5 , human herpes simplex virus , Dengue Virus type 2  and echovirus E1 (one case).The date of collection of specimens that were test negative using the routine viral panel assay, and their NGS viral detection results, are plotted by time Fig. . The varCategory 1 The virus was in the diagnostic panel but locally circulating strains differed at diagnostic primer/probe sites. This is remedied by updating the diagnostic panel with locally appropriate primers, as being done recently2. Category 2 The virus was detected but below the cut-off for a positive diagnosis. The current cut-off for positive diagnosis is PCR Ct < 35.0; yet the assays did not show Ct-values of negative diagnoses, hence limiting the interpretation of findings here. Further studies are needed to determine assay sensitivities to update positive/negative cutoffs. Category 3 The virus was not in the diagnostic test panel. Due to practical constraints, it is not feasible to include all potential pathogens in a diagnostic panel, however findings from NGS analyses may support the decision to modify diagnostic panels accordingly. A limitation of this NGS approach is the threshold below which a virus does not yield identifiable sequences. Although 30.5% of the samples returned a viral diagnosis, 69.5% failed to yield a classifiable viral sequences. We expect that future improvements in NGS methods will increase the fraction of new diagnoses allowed by these methods.Respiratory infections are a leading cause of morbidity and mortality worldwide, sensitive and accurate viral diagnostics are crucial for guiding clinical care. In this study, we investigated clinical respiratory samples from a single location in Kenya that had failed to return a diagnosis with the local PCR diagnostic panel. We used randomly primed (unbiased) metagenomic sequencing to increase the viral detection potential. The objectives of this study were twofold. Firstly, we were interested in the number of missed virus diagnoses in respiratory cases. Secondly, we sought respiratory infections in Kilifi whose agents were not included in the routine screening panel. Here we have demonstrated the utility of direct deep sequencing of clinical respiratory samples to identify virus genomes circulating in a resource-limited country. When applying this simple strategy of random-primed viral NGS in respiratory samples testing negative in local diagnostic panels, viral sequences were identified and the approach revealed several categories of missed diagnostics. 18. Co-infections with multiple viruses were also detected which may also account for disease severity. Finally, the methods yielded complete or nearly complete genome sequences for respiratory viruses circulating in Kilifi providing a valuable sequence resource for improving local PCR diagnostic assays.The unexpected/not-tested viruses , providing additional possible causative agents explaining the symptoms and could help in the local guidelines and policy for disease management and practice. Common viruses were found that had been missed by diagnostics . In many cases these detected virus sequences showed nucleotide changes in the primer and/or probe target sites that may account for the missed detection. Although rhinoviruses are the most common virus diagnosis in the Kilifi setting, they were frequently missed by the diagnosticsAlthough viral NGS would be expensive to apply for all diagnostics, the data from this study can inform an optimum pace of applying agnostic viral NGS to improve local diagnostics. The concept of the idea is illustrated in Supplementary Fig. The data and analyses presented here provide a description of circulating respiratory viruses from two cohorts  from one region in coastal Kenya. Albeit small sample size and from a single location, the study included patients with a wide range of symptoms ranging from runny nose, sneezing to severe and very severe pneumonia, with all case types failing to yield a diagnosis for an aetiological pathogen. The study setting and methodologies, i.e. the combination of agnostic viral NGS on samples that had also been subjected to the local viral diagnostic panel, are the strength of the study. This combination allowed us to make important conclusions about the number and type of viruses missed by the local viral diagnostic panel and therefore provides useful information for improving local viral diagnostics.5 and a household cohort investigating Who-Acquires-Infection-From-Whom (WAIFW)9 from Kilifi, coastal Kenya. The hospital surveillance enrolled paediatric patients presenting with severe or very severe pneumonia based on clinical symptoms as previously defined5, and included children aged 1 day to 59 months and excluded babies with neonatal tetanus. For the KCH surveillance, naso- and oropharyngeal swab (NP/OP) samples were collected as soon as possible after hospital admission as previously described19. Ninety-five samples which had tested negative for the 15 respiratory viruses ses see “” section9. Ninety-five samples from household members with symptoms of upper respiratory tract infection, which had tested negative for viral pathogens diagnosis, were chosen and represent the mild spectrum of respiratory infections in this study.For the household cohort, members of households in a rural coastal in Kilifi were enrolled and an NP swab was collected from all members irrespective of respiratory symptoms at regular twice-weekly visits from December 2009 to June 201020: respiratory syncytial virus (RSVA and B), influenza virus A, B, and C, human rhinovirus (HRV), human coronavirus , adenovirus (AdV), human parainfluenza virus (HPIV1–4), and human metapneumovirus (HMPV). Primers, probes and target genes used for the PCR assays are summarised in Supplementary Table 5. The multiplex real-time PCR with or without the IFAT assay is referred to as “the standard diagnostic panel” throughout the manuscript.All samples were screened by multiplex real-time PCR for 15 respiratory virus targets21. Briefly, the method includes centrifugation and DNase treatment to remove free non-encapsidated DNA, reverse transcription with non-ribosomal random hexamers avoiding rRNA targets followed by sequencing on Illumina HiSeq 2500, generating 2–3 million 250 nt paired-end reads/sample.Total nucleic acid extraction and dsDNA conversion were performed as previously described22) were de novo assembled (SPAdes v.3.1023). Virus contigs were identified with UBLAST24 using virus family protein databases. Final quality control of genomes included checking open reading frames (ORFs), and comparison with reference sequences retrieved from GenBank. Stringent criteria for calling a sample positive for required greater than or equal to 1000 nt .Quality controlled reads  with 100 pseudoreplicates. Bootstrap values of ≥ 70% were considered statistically significant and were shown for major nodes. Tree was visualised in FigTree v.1.4.3 (http://tree.bio.ed.ac.uk/software/figtree). The tree was mid-point rooted for clarity and horizontal branch lengths were drawn to the scale of nucleotide substitutions per site.Global reference sequences were retrieved from GenBank, coding regions from reference and assembled genomes were extracted, and aligned using MAFFTThe study was approved by the Kenyan Medical Research Institute Scientific and Ethics Review Unit (KEMRI-SERU) and the Coventry Research Ethics Committee (United Kingdom), and all methods were performed in accordance with the relevant guidelines and regulations by KEMRI-SERU and the Coventry Research Ethics Committee. Written informed consent was obtained for all eligible participants before sample collection. For children (< 18 years), informed consent was given by parents or guardians.Supplementary Legends.Supplementary Information 1.


The epigenetic state of chromatin is altered by regulators which influence gene expression in response to environmental stimuli. While several post-translational modifications contribute to chromatin accessibility and transcriptional programs, our understanding of the role that specific phosphorylation sites play is limited. In cancer, kinases and phosphatases are commonly deregulated resulting in increased oncogenic signaling and loss of epigenetic regulation. Aberrant epigenetic states are known to promote cellular plasticity and the development of therapeutic resistance in many cancer types, highlighting the importance of these mechanisms to cancer cell phenotypes. Protein Phosphatase 2A (PP2A) is a heterotrimeric holoenzyme that targets a diverse array of cellular proteins. The composition of the PP2A complex influences its cellular targets and activity. For this reason, PP2A can be tumor suppressive or oncogenic depending on cellular context. Understanding the nuances of PP2A regulation and its effect on epigenetic alterations can lead to new therapeutic avenues that afford more specificity and contribute to the growth of personalized medicine in the oncology field. In this review, we summarize the known PP2A-regulated substrates and potential phosphorylation sites that contribute to cancer cell epigenetics and possible strategies to therapeutically leverage this phosphatase to suppress tumor growth.  Aberrant epigenetic regulation of the cancer genome contributes to a wide range of phenotypes, including therapeutic resistance and cellular plasticity . In normIn addition to specific DNA sequences that influence gene transcription, there exists a higher order structure to the genome that provides an additional layer of regulation to maintain cellular homeostasis. The nucleosome comprises DNA wrapped around a histone octamer , which shields the associated DNA from the transcriptional machinery. Reversible epigenetic marks, such as DNA methylation or posttranslational modifications (PTMs) on histones, allow for dynamic changes in the genome without altering the DNA sequence. In response to repressive epigenetic marks, histones will condense to form heterochromatin, or ‘silent’ chromatin regions. Active epigenetic marks help direct remodeling complexes to specific sites of DNA to displace histones and expose critical coding regions, providing additional security that only genes in euchromatic, ‘active’ chromatin regions are readily expressed. Densely packed regions of chromatin are moved to the nuclear lamina to make room for the transcriptional machinery within areas of open chromatin. This dynamic spatial organization, or 3D architecture, generates topologically associated domains (TADs) within the nucleus. During tumorigenesis, the epigenetic regulation of these various chromatin states is frequently altered, significantly contributing to cancer phenotypes and therapeutic response . HoweverThere is a diverse array of cellular complexes that balance the deposition, reading, and removal of epigenetic marks to rapidly alter gene expression based on changes in the cellular environment. These epigenetic modifiers can be divided into three main categories: ‘readers’, ‘writers’ and ‘erasers’. Epigenetic ‘writers’ and ‘erasers’ deposit or remove epigenetic marks, such as methylation, on DNA and histones ,8. EpigeKinase inhibitors have been a primary focus for anti-tumor therapeutics contributing to the rise in personalized medicine . HoweverThe function of PP2A and its contribution to tumorigenesis is highly dependent on the composition of the PP2A holoenzyme. Here, we review the complex mechanisms by which PP2A regulates the epigenetic state of cancer cells and potential ways to leverage this activity for anti-cancer therapeutics.The loss of normal chromatin regulation leads to irregular nuclear morphologies, heterochromatin redistribution and deregulated transcriptional programs, emphasizing the importance of understanding how chromatin states are dynamically regulated . ChromatHeterochromatic regions are formed in response to repressive histone modifications, such as histone H3 lysine 9 di- and trimethylation (H3K9me2/me3) ,33. Theset al. demonstrated that PRR14 contains a conserved short linear motif (SLiM) consistent with PP2A-B56 substrates. This SLiM motif drives the association of PRR14 and PP2A complexes containing B56α (PP2A-B56α)  ,38. PRR12A-B56α)  37,39).,39.et alAdditionally, direct phosphorylation of Lamin proteins during mitosis promotes depolymerization and breakdown of the nuclear lamina so that cells may properly undergo cell division . Both PPIn addition to anchoring heterochromatic regions, Lamins also contribute to the distribution of nuclear pore complexes (NPCs) across the membrane and the transcriptional regulation of genes associated with these structures. Although the majority of DNA associated with the nuclear lamina is heterochromatic, the regions surrounding the NPC are commonly euchromatic and highly transcribed . NPC-assTogether, these studies support a model where CIP2A-mediated inhibition of PP2A at the nuclear periphery to facilitate the oncogenic activities of MYC. However, the PP2A subunits that are involved in the process and the contribution of Lamin A/C phosphorylation to association with oncogenic phenotypes need to be further explored.Phosphorylation of histone H3 (H3) at the serine 10 residue (pH3S10) provides a docking site for chromatin modifiers and increases the acetylation of neighboring residues . This PTPP2A is reported to alter the activity of chromatin remodeling complexes that regulate or ‘read’ histone epigenetic marks in addition to the direct dephosphorylation of histones. Aberrant regulation of PP2A holoenzyme composition and activity alters the precise signaling that methylation- and acetylation-associated complexes provide, further contributing to oncogenesis by indirectly influencing euchromatic region accessibility.Histone acetylation primarily results in the relaxation of the chromatin and increased transcription of nearby genes. Acetylation marks are added and removed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively, which function in a coordinated effort to maintain normal transcriptional programs. While increased acetylation is often associated with cancer, elevated levels of HDAC activity can similarly contribute to tumor phenotypes by silencing key tumor suppressor genes, such as p53 ,63. DespPP2A also regulates the activity of lysine acetylation ‘readers’, including the Bromodomain and Extraterminal (BET) protein, Bromodomain-containing 4 (BRD4). BRD4 accumulates at euchromatic regions to promote gene transcription through the recruitment of transcription factors to the transcription start site or as a part of super-enhancer complexes . Phosphoet al. unexpectedly demonstrated that rather than global transcriptional repression, the BET inhibitor, JQ1, only modified the expression of a limited number of genes, indicating that specific genes displayed unique susceptibility to BET inhibition. Of the affected targets, the MYC super-enhancer was acutely reliant on BRD4-dependent transcriptional activation  complexes play an important role in suppressing gene transcription by removing acetylation marks and promoting formation of heterochromatin. Given that deregulation of HDAC activity can contribute to either tumor suppressive or oncogenic signaling, proper HDAC nuclear localization is extremely important for cellular homeostasis . Class IPP2A-mediated regulation of HDAC complexes greatly depends on the composition of the PP2A holoenzyme . In contHDAC4 signaling contributes to several cancer phenotypes, including increased proliferation and suppression of differentiation transcriptional programs . AlthougRegulation of HDAC4 by PP2A has also been shown to impact the differentiation state of fibroblasts . ReprogrHistone methylation marks are extremely diverse in function and can be associated with promotion or inhibition of gene transcription. The role of each methylation mark depends on the genomic site, the presence of other neighboring histone PTMs or epigenetic marks, as well as the number of methyl groups deposited on any given residue  . SimilarWhile methylation of lysine residues (K) is more common, the field of arginine (R) methyltransferases has significantly expanded within the past decade. There are three subclasses of protein arginine methyltransferases (PRMTs): (i) asymmetric dimethylarginine (ADMA), (ii) symmetric dimethylarginine (SDMA) and (iii) monomethyl arginine (MMA) . All PRMPRMT1 is the predominant ADMA methyltransferase, accounting for approximately 85% of total arginine methyltransferase activity . PRMT1 pPRMT5 is the predominant SDMA methyltransferase and is generally associated with gene repression . There iIn addition to histones, cytosine nucleotides can also be directly methylated. Methylated cytosine results in the eventual formation of large stretches of CpG islands, or areas of dense DNA methylation, normally found at promoters of actively repressed genes. Reversal of methylated cytosine is a process that requires multiple oxidative reactions by ten-eleven translocation (TET) proteins and conversion to an abasic site that can be recognized for base excision repair . The TETet al. earlier this year  has been used as a targeted approach to identify not only if two proteins interact (phosphatase-substrate) but the location of the complex within the cell. This additional spatial information is invaluable for dissecting potential subcellular domains of phosphatase function  and dynamic shifts in protein localization or PP2A holoenzyme composition in response to perturbations such as oncogenic mutation. To decipher transcriptional regulatory complexes, chromatin immunoprecipitation with selective isolation of chromatin-associated proteins (ChIP-SICAP) is now being used to isolate both protein complexes and their associated DNA in the As we begin to unravel the context-specific roles of PP2A in the regulation of epigenetic and transcriptional mechanisms, new compounds could be developed to capitalize on the unique functions of individual PP2A B subunits. Already, compounds are emerging that preferentially activate unique PP2A complexes (SMAPs and iHAP1), suggesting that eventually we may be able to tailor therapeutics to specific PP2A-dependent cancer phenotypes . Since t