Elevated concentrations of serum phosphate are linked with progression and increased case fatality rate in animals and humans with chronic kidney disease. Elevated concentrations of serum phosphate can be a risk factor for development of renal and cardiovascular diseases or osteoporosis in previously healthy people. In rodents, an excess intake of dietary phosphorus combined with an inverse dietary calcium : phosphorus ratio (<1 : 1) contributes to renal calcification. Renal injury also has occured in cats fed experimental diets supplemented with highly soluble phosphate salts, especially in diets with inverse calcium : phosphorus ratios. However, not all phosphorus sources contribute similarly to this effect. This review, which focuses on cats, summarizes the published evidence regarding phosphorus metabolism and homeostasis, including the relative impact of different dietary phosphorus sources, and their impact on the kidneys. No data currently shows that commercial cat foods induce renal injury. However, some diets contain high amounts of phosphorus relative to recommendations and some have inverse Ca : P ratios and so could increase the risk for development of kidney disease. While limiting the use of highly soluble phosphates appears to be important, there are insufficient data to support a specific upper limit for phosphate intake. This review also proposes areas where additional research is needed in order to strengthen conclusions and recommendations regarding dietary phosphorus for cats. Elevated concentrations of blood phosphate or maladaptive activation of hormonal systems involved in regulating phosphate could have direct toxic effects on the kidneys and cardiovascular system.This review summarizes the published evidence regarding P metabolism and homeostasis, including how these factors affect the kidneys. This review further notes the limitations and gaps in the existing evidence, and proposes areas where additional research is needed in order to strengthen conclusions and recommendations regarding dietary P for cats.2Dietary P is absorbed in the small intestine via 2 general mechanisms: sodium‐dependent active absorption (primary route), and concentration dependent paracellular diffusion (secondary route) in humans and rodents. Active absorption of P is up‐ or down‐regulated depending on the animal's needs and various regulating factors, while paracellular diffusion is more dependent on the total amount consumed.Increased dietary protein appeared to enhance renal Pi excretion and decrease Pi retention in both adult and growing cats, so P requirements might be dependent on dietary protein content.2PO4) and monopotassium phosphate (KH2PO4) significantly increased sPi and PTH compared to similar amounts of P from poultry meal  while another study showed that the effects of calcium diphosphate (Ca[H2PO4]2) were intermediate between phosphorus from poultry meal and monosodium phosphate .Dietary P can be intrinsic to ingredients, such as meats, bone meals, and grains, or be added as inorganic sources such as mined phosphate rock, phosphoric acid, or various phosphate salts. Table . PhosphoMeta‐analysis of digestion trials in dogs and cats estimated the average true digestibility of P to be 17% in dogs and 31% in cats.3) increases, intestinal absorption of dietary P and urinary excretion of Pi are both reduced in a linear manner.Dietary Mg is another macromineral that interacts with Ca and P. In cats, as dietary Mg . As GFR decreases in the face of CKD, the amount of filtrate decreases. Likewise, for a given GFR, the sPi affects the amount of Pi entering the tubules. Thus, as sPi increases, the amount of Pi entering the tubules increases despite the reduced GFR. Once in the renal tubules, reabsorption or excretion is controlled by PTH, 1,25D3, and FGF23, as noted above. However, these also are impacted by CKD. Both FGF23 and PTH are increased in CKD which result in reduced renal tubular reabsorption of Pi and greater urinary Pi excretion, reduced activation of 1,25D3 and reduced intestinal P absorption,3Hyperphosphatemia contributes to ongoing damage in the face of existing kidney disease, and dietary P restriction can help reduce this ongoing damage.Female rats are predisposed to development of nephrocalcinosis, or calcification of kidney tissue especially at the corticomedullary junctions. This form of kidney disease is responsive to dietary P and to the dietary Ca : P ratio.3PO4) for a large portion of the P  and also had an inverse Ca : P ratio, both of which can alter bioavailability and renal effects of P.In dogs with induced CKD, restriction of dietary P reduces the associated morbidity and improves survival.Evidence in people has been accumulating that elevations in sPi can be a risk factor for development of renal disease, cardiovascular disease and increased mortality even in patients with apparently normal kidney function.A small study in pet dogs and cats suggests a positive correlation between high P intake (assessed through dietary questionnaire) and CKD in cats, but not in dogs.High Pi concentrations alter vascular and endothelial function and alter the nitric oxide pathway in in vitro studies.4Although there is currently no evidence that P in commercial cat foods induces renal disease in healthy cats, high “ash” content in food might be a risk factor for developing CKD.2PO4‐2H2O). The Ca : P ratios ranged from 1.8 : 1 down to 0.3 : 1.Studies in healthy cats show an impact of dietary P, or inverse Ca : P ratios, on markers of kidney function.4[PO4]2) and monosodium‐(NaH2PO4) phosphates.In the second study, healthy cats developed glucosuria, microalbuminuria and showed significantly decreased creatinine clearance within 28 days of being fed a diet containing approximately 3 g P/Mcal ME (1.6% of diet DM) with approximately 2/3 provided as a combination of monocalcium  to deliver 4.78 g P/Mcal ME (1.93% diet DM) with a Ca : P ratio of 0.6 to a control diet providing 1.23 g P/Mcal ME (0.52% diet DM) and Ca : P at 1.1.A third study compared a diet supplemented with sodium dihydrogen phosphate  with supplemental inorganic P from mono‐ and di‐basic sodium phosphates, and confirmed significantly greater uptake, along with increases in sPi and uPi in cats fed the more digestible inorganic P.5P3O10).A different study design was used in series of acute kinetic trials in cats fed diets with 2 different inorganic sodium phosphate sources, SDHP and sodium tripolyphosphate ; reduced accumulation of dental tartar ; and, processing aids to ensure optimal cooking and texture . Among commercial cat foods in the United States, the most commonly added phosphorus sources for wet foods include tricalcium phosphate, dicalcium phosphate, and sodium tripolyphosphate, while phosphoric acid and dicalcium phosphate are the most commonly added sources of phosphate in dry cat foods Table .2PO4) may affect indicators of renal health in cats . More research is needed to clarify potential risk.”United States pet food regulations address both nutrient content of foods and ingredients used. Table United States pet food regulations require all ingredients, including specific sources of P, to be listed individually in descending order by weight but this is not required for European pet foods. Furthermore, neither US nor European regulations require that total amounts of P or Ca be listed on commercial pet food labels. Many cat foods in the United Kingdom are not in compliance with published nutritional guidelines, and more relevant to the topic of this paper, about 10% have inverse Ca : P ratios (below 1 : 1).6Based on currently available evidence, it is clear that excess phosphorus, especially when provided as highly soluble inorganic phosphate salts, can be damaging to kidneys. This effect appears to be more pronounced in foods with an inverse Ca : P ratio. What is not known is the impact of various phosphate salts which vary in digestibility and physiological effects. Safe upper limits have not been established for the various Pi salts. Adverse effects can occur with high Pi diets in relatively short term studies, but might lower amounts be detrimental over longer periods or do physiological adaptations occur to protect against these effects and, if so, do individuals vary in their ability to adapt? Other dietary factors, such as the presence of phytates, fibers, other minerals, differences in base excess and differences in the base diet matrix, also may alter the impact of dietary P. No data currently shows that commercial diets induce renal injury. However, some commercial diets contain high amounts of phosphorus relative to recommendations and some have inverse Ca : P ratios, so could increase the risk for development of CKD.It is clear that more research is needed. Until more evidence is available, the authors recommend avoiding diets with a Ca : P less than 1 : 1. Postprandial increases in serum Pi and PTH can occur when highly soluble sodium phosphates are added to diets in amounts equal to or greater than 0.5gP/Mcal, despite a Ca : P ratio above 1 : 1. However, similar results do not occur with other inorganic phosphates, such as calcium phosphates, and many inorganic phosphate sources have not yet been evaluated in this manner. While limiting the use of highly soluble phosphates, such as sodium‐ or potassium‐phosphates, appears to be important, there are insufficient data to support a specific upper limit for inorganic or soluble phosphates at this time. Among the additional research needed are studies to identify sensitive markers of renal injury; to identify risk, if any, from current commercial products; and to generate more information about the kinetics, impact of different phosphorus sources, and the physiological adaptations to long term feeding of highly bioavailable phosphates.Expenses related to the development of the manuscript  were offset by the sponsors, the Purina Institute and the Waltham Petcare Science Institute. Laflamme is a consultant for Purina. Backus receives support for his nutrition program from a Purina endowment, has consulted for both sponsors, and has received other support for his nutrition program from both sponsors and Hill's Pet Care. Butterwick and Czarnecki‐Maulden are employed by the sponsors. Elliott has served as a consultant for Waltham and has received research funding from Royal Canin. Fascetti serves on an advisory council and is a frequent speaker for the Purina Institute, and has received support for her nutrition program from both sponsors and Hill's Pet Care.Authors declare no off‐label use of antimicrobials.Authors declare no IACUC or other approval was needed.Authors declare human ethics approval was not needed for this study.


Arteriovenous malformation(AVM) have long-term “blood stealing” characteristics, which result in complicated hemodynamic features. To analyze the application of intraoperative indocyanine green angiography with FLOW 800 software in AVM surgeries.Data on 17 patients undergoing surgery with ICG fluorescence were collected in Beijing Tiantan Hospital. To analyze the hemodynamic features of AVM and the influence on the peripheral cortex of AVM resection, we assessed the following hemodynamic parameters: maximum intensity, slope of rise, time to half-maximal fluorescence, and transit time from arteries to veins.P < 0.05). The transit times in AVMs were significantly shorter than those in normal peripheral vessels (P < 0.05). After AVM resection, cerebral flow increased in the cortex, and local cycle time becomes longer, although the differences were not significant (P > 0.05).In the 17 superficial AVMs studied, the time-delay color mode of the FLOW 800 software was superior to the traditional playback mode for identifying feeding arteries, draining veins, and their relation to normal cortical vessels. The maximum fluorescence intensity and slope of the ICG fluorescence curve of feeder arteries and draining veins were higher than those of normal peripheral vessels (Hemodynamic parameter analysis provided quality guidance for the resection of AVMs and could also be used in estimating changes in blood flow in the local cortex to identify abnormal hyperperfusion and residual nidus. Arteriovenous malformations (AVMs) are responsible for about 2% of all hemorrhagic strokes, mainly in children and young adults , 2. DespIntraoperative indocyanine green (ICG) videoangiography is used widely in neurosurgery and has proven a useful addition to neurovascular surgery –6. FLOW In this study, we analyzed hemodynamic changes in AVM nidus and normal peripheral vessels during surgery using ICG videoangiography and the FLOW 800 software.The study protocol and written informed consent form were reviewed and approved by the Institutional Review Board of Beijing Tiantan Hospital , which is affiliated with Capital Medical University  (KY 2017-012-02).We enrolled 17 patients who were admitted to our department with AVM during March to November 2016  with peak spectral absorption at 805 nm and peak emission at 835 nm [All operations were performed using two microscopes available in our department . Both microscopes had near-infrared video integration and were able to perform ICG videoangiography.Under illumination with a near-infrared light source (excitation range 700–780 nm), real-time microscope images were recorded. An optical filter that allowed only fluorescence in the ICG emission range (820–900 nm) was used. To obtain optimum illumination, we set the standards for AVM resection as follows: the microscope diaphragm was automatically adjusted to a wide setting; the illuminated field diameter was set in the middle position; the illumination intensity was set to 50%; the recommended working distance of the scope was set at 300 mm; and the Pentero microscope zoom was set to 5 ×.ICG fluorescence angiography clearly showed the AVM nidus. Feeder arteries and draining veins were mainly chosen as the hotspots. We selected three to five feeder arteries and draining veins, along with peripheral cortical arteries, as the hemodynamic parameter collection points using the FLOW 800 software.FLOW 800 is an analytic color visualization tool that can evaluate the fluorescence video sequences obtained by microscope-integrated ICG fluorescence angiography using INFRARED 800. It provides an objective evaluation of results, visually and in different colors. Using this visualization tool, the ICG transit curve intensities were recorded, and maps of maximal fluorescence intensities were saved. Then, two-dimensional visual maps (including color maps) of the AVM were created according to maximal fluorescence intensities or time to half-maximal fluorescence. The intensity diagram function can provide a variable shape that reflects variation in the blood flow of the feeders, drainers, and peripheral vessels before and after AVM resection. Related parameters were defined as follows: time to half-maximal fluorescence was defined as the time required for the ICG fluorescence intensity to reach 50% of the maximum value (T1/2 peak); transit time was defined as the time required for blood to flow from artery to vein (arteriovenous transit time); rise time was defined as the interval between 10% and 90% of the maximum signal; and cerebral blood flow index slope was defined as the ratio of maximum fluorescence intensity to rise time.The indication for each ICG video during the AVM resection was recorded. As recommended by Hanggi et al. , 9, the t test. Two-tailed P values < 0.05 were considered statistically significant.Values are presented as the mean ± standard deviation. The statistical analysis was performed using the Statistical Package for the Social Sciences ver. 16.0 . The mean measurements of the computed tomographic angiography (CTA) and three-dimensional model were compared using one-way analysis of variance followed by Fisher’s least significant difference test. Comparisons between two groups (survey scores) were achieved using Student’s Our study enrolled 17 AVM patients who were judged suitable for surgical resection surgery under general anesthesia. The demographic characteristics of the patient population are listed in Table A total of 51 ICG fluorescence angiographies were performed; 15 patients received angiography three times, 1 received it two, and 1 patient received angiography four times. Blood pressure was maintained at 100–120 mmHg (105 ± 12 mmHg). Fourteen patients underwent digital subtraction angiography (DSA) or CTA after the operation, and all tests showed that the AVM was totally removed.No side effects or adverse reactions were noted after ICG administration in any of the patients.P < 0.05; Figs. In the 17 superficial AVMs, the time-delay color mode provided by the FLOW 800 software proved more useful than the traditional playback mode in identifying feeding arteries, draining veins, and their relations to normal cortical vessels. The transit times in AVMs were significantly shorter than those in normal peripheral vessels (P < 0.05), indicating that AVMs exhibited hemodynamic characteristics of high flow and high perfusion  was significantly earlier than that for cortical arteries (T1/2 peak 22.59 ± 3.13 s), which could serve as a basis for identifying feeder arteries. The slope of arterial feeders (144.95 ± 38.08 AI/s) was significantly greater than that of normal cortical arteries . This observation could be helpful in identifying draining veins , slope of the ICG fluorescence curve (178.66 ± 15.09), and T1/2 peaks (20.79 ± 3.42 s) of draining veins were higher than those of normal peripheral vessels (7 ± 90.41P < 0.05). The microvascular transit time (MVTT) of the cortical arteries was 2.08 ± 1.42 s before and 2.59 ± 1.21 s after resection. The cerebral flow in the cortex increased, and the local cycle time became longer, although these differences were not significant . Nonetheless, the results showed the tendency of AVM removal to affect the cortical veins.Although we found that cerebral flow in the cortex increased and local cycle time became longer, these differences were not significant . The results showed that cortical blood perfusion was increased; in some local areas, hyperperfusion was noted. The delayed cycling time showed that AVM resection may have caused local cortical venous reflux.In a previously published hemodynamic study, Ng et al. showed that ICG videoangiography was useful in different phases of AVM surgery . FurtherAfter AVM resection, the slope of ICG fluorescence in the peripheral cortical arteries was significantly increased (from 69.20 to 106.63), the maximum fluorescence intensity was enhanced, and image develop of cortical arteries was earlier than before surgery. This showed that cortical perfusion was increased after the AVM resection; indeed, some parts exhibited hyperperfusion. However, the time of circulation was prolonged, suggesting venous reflux due to obstruction in some parts of the cortex, a phenomenon termed “venous overload.” Wilson et al. proposed that venous overload was caused by thrombosis in a draining vein due to clogging by an AVM remnant . Our resWe compared the T1/2 peak fluorescence rate, slope, and maximum fluorescence intensity of the draining and cortical veins. The results showed that the timing of the T1/2 peak fluorescence rates was earlier for the draining than for the cortical veins, and the slope and maximum fluorescence intensity of the draining veins were greater than those of the cortical veins. These findings may prove useful in identifying abnormal draining veins. The real-time feedback regarding blood flow provided by FLOW 800 could help the surgeon to determine or modify the operative strategy for a safe and complete AVM resection .A combination of intra- and postoperative DSA is regarded as the gold standard for identifying AVM remnants after surgery, especially in small and deep AVM surgery . But disIntraoperative ICG videoangiography combined with analysis of hemodynamic parameters obtained by the FLOW 800 software is a convenient and effective method for evaluating the hemodynamic features of superficial AVMs by identifying the feeding arteries, draining veins, crossing arteries, and cortical vessels, and to provide quality guidance for AVM resection. Furthermore, hemodynamic parameter analysis can be used for estimating changes in blood flow in the local cortex, thereby identifying residual nidus and vessels contributing to abnormal hyperperfusion.


Corn is the main feed ingredient used in swine diets as an energy source due to its abundant production and nutrient contents. In South Korea, most of the corn for animal diets depends on import from other countries—more than 7.5 million tons per year. Thus, there is a need to find alternative ingredients to substitute corn in pig diets. Although there are variations depending on the degree of milling, brown rice has similar or better nutrient contents compared to corn. In addition, it is known to have excellent digestibility due to its smaller starch structure and granule size and less non-starch polysaccharides and anti-nutritional factors than corn. As a result of evaluating the effects of replacing corn with brown rice in pig diets, changes in gut microbiota were observed when corn was replaced with brown rice for a long time, but there were no differences on growth performance and carcass characteristics. Therefore, it has been confirmed that brown rice can replace corn in swine diets and the use of brown rice as a pig feed ingredient may be the basis for increasing feed self-sufficiency and enabling a stable feed supply.The present study was conducted to evaluate the effects of replacing corn with brown rice on growth performance, nutrient digestibility, carcass characteristics, and gut microbiota of growing and finishing pigs. A total of 100 growing pigs  were randomly allotted to 4 dietary treatments  in a randomized complete block design (block = BW) as follows: corn-soybean meal basal diet (CON) and replacing corn with 50% (GBR50), 75% (GBR75), and 100% (GBR100) of ground brown rice. Each trial phase was for 6 weeks. During the growing period, there were no differences on growth performance and nutrient digestibility among dietary treatments. Similarly, no differences were found on growth performance, nutrient digestibility, and carcass characteristics of pigs during the finishing period among dietary treatments. As a result of the beta diversity analysis, microbial populations were not clustered between CON and GBR100 during the growing phase, but clustered into two distinct groups of CON and GBR100 during the finishing phase. In conclusion, brown rice can be added to the diets of growing-finishing pigs by replacing corn up to 100% without negatively affecting growth performance of the pigs; additionally, this may have an effect on changes in pig intestinal microbiota if continued for a long time. Corn is the main feed ingredient used in swine diets as an energy source due to its abundant production, nutrient contents, and relatively long storage. Rice is the world’s third most grown staple crop providing 50 percent of the world’s calories along with corn and wheat . Since rAlthough the quality of nutrient contents varies depending on the degree of milling, except for whole grains (paddy rice), rice contains more starch than corn  and its The mammalian gut intestinal tract (GIT) has diverse and active microbial communities that provide important signals for the development of the immune system and for functions such as digesting and absorbing nutrients . Diet coSeveral studies indicate that brown rice can completely replace corn in swine diets for weanling and growing pigs without negative impacts on growth performance and apparent digestibility of nutrients ,15,16. HThe protocol of this experiment was reviewed and approved by Institutional Animal Care and Use Committee at the Chungnam National University, Daejeon, South Korea . This experiment was conducted at the Animal Research Center of Chungnam National University, Cheongyang, South Korea.A total of 100 growing pigs  were used in this experiment. These pigs were randomly allotted to 4 dietary treatments  in a randomized complete block design (block = BW). After finishing the study for growing period, pigs were randomly relocated within groups and then the study for finishing period was conducted. Dietary treatments were growing and finishing diets based on corn-soybean meal basal diet (CON) and three additional diets formulated by replacing corn with 50% (GBR50), 75% (GBR75), and 100% (GBR100) of ground brown rice. The brown rice used in this experiment was ground with an average particle size of 0.5 mm (0.4–0.6 mm). The pigs were fed respective dietary treatments for 6 and 6 weeks for the growing and finishing periods, respectively. The dietary treatments were formulated to meet or exceed the nutrient requirement of growing and finishing pigs  and had Pigs were individually weighed at 1 (the first day), 42, and 84 d to measure average daily gain (ADG). Feed intake was recorded for each pen to calculate average daily feed intake (ADFI) and feed efficiency (G:F) for growth performance of growing and finishing periods. At the beginning of the last week of each period, 0.2% chromic oxide as an indigestible marker was mixed into each dietary treatment and the diets were fed to pigs during the last week of each period. For the periods, initial 4 days were adaptation days and feces were collected with anal massage method for 3 consecutive days. The fecal samples were collected from randomly selected 1 pig in each pen and stored at −80 °C for analysis to determine apparent total tract digestibility (ATTD) of nutrients. Three pigs from CON and GBR100 were randomly selected to collect feces on the last day of each experimental phase (d 42 and 84) and stored at −80 °C for analysis to verify their microbiota changes by pyrosequencing analysis. At the end of the experiment, the pigs were transferred to a local commercial slaughterhouse and treated with conventional process. The live BW of pig in each treatment was recorded just before slaughter. The hot carcass weight (HCW) was recorded and dressing percentage was calculated by comparing final BW and HCW. Then, carcasses were chilled at 2 °C for 24 h. At 24 h post-mortem, the right-side loin was taken between the 10th and 11th ribs to measure back fat thickness according to National Pork Producers Council .Diet and frozen fecal samples were oven-dried at 135 °C for 2 h and then finely ground before chemical analysis. Prepared diets and fecal samples were analyzed for dry matter  , nitrogeTotal DNA representing the fecal microbiota was extracted from 300 mg of fecal contents per sample using QIAamp DNA Stool Mini Kit  following manufacturer’s instructions. DNA concentration and quality were assessed using NanoDrop ND-1000 spectrophotometer . Genomic DNA was stored at −70 °C until further analysis.The V4 region of the 16S rRNA genes was amplified by PCR using featured primers as listed previously . The 16SRaw sequence data were quality filtered using the Mothur software to remove low-quality sequences . Sequencp < 0.05 and 0.05 ≤ p < 0.10, respectively.Data were analyzed using the PROC MIXED of SAS  in a randomized complete block design (block = BW). Experimental unit was the pen. The statistical model for growth performance, ATTD of nutrients, and carcass characteristics of pigs included effect of dietary treatments as a fixed effect and initial BW as a random effect. Pair-wise comparisons were performed among dietary treatments when main effects of dietary treatments were observed. Results are expressed as mean ± standard error of mean (SEM). Statistical significance and tendency were considered at The dietary treatments were formulated to have similar concentrations of CP and GE. The analyzed concentrations of CP and GE in the diets were 19.43–20.64% and 4325–4446 kcal/kg, respectively, for growing pigs and 16.78–18.54% and 4275–4486 kcal/kg, respectively, for finishing pigs .There were no differences on ADG, ADFI, and G:F of pigs during growing phase among the dietary treatments . SimilarNo differences were observed on ATTD of DM, energy, and CP for growing and finishing pigs among dietary treatments . There were no differences on carcass characteristics such as live BW, HCW, dressing percentage, and back fat thickness among dietary treatments .After quality filtering, the average number of sequence reads obtained from growing pigs were 181,852 ± 19,196 (mean ± SD) for CON and 186,395 ± 16,116 for GRB100 . The meaThe PCoA plot visually confirmed a distinct separation of microbial communities and characterized the differences of gut microbial communities between CON and GBR100 groups for growth phases . MicrobiTaxonomic classification of the bacterial 16S rRNA genes at phylum and genus levels are shown in Lactobacillus was the most abundant genus accounting for more than 40% of the total sequences for pigs in both treatment groups  in pigs fed GBR100 (31.80%) compared to that in pigs fed CON (18.70%). On the other hand, the relative abundance of phylum Firmicutes was significantly higher (p < 0.05) in CON pigs (63.11%) compared to that (44.66%) in GBR100 pigs  in GBR group (16.96%) compared to that in CON group  and Streptococcus (6.79% vs. 17.32%) were less abundant (p < 0.05) in finishing pigs fed GBR100 compared with those in finishing pigs fed CON . The Oscilibacter and Roseburia are bacteria that produce organic acids by fermenting carbohydrates [Yang et al.  previousity pork ,62. Parkity pork  showed tity pork  and the hydrates ,63. In thydrates . The bacterial 16S rRNA gene sequencing was used to compare the fecal microbial compositions between treatment groups. According to the alpha diversity analyses, there were no significant changes of the gut microbial evenness and richness between treatment groups at the same sampling time point. However, the PCoA plot showed the differences of the gut microbial memberships and their relative abundances between CON and GBR100 groups for finishing pigs. Based on the PCoA plot, microbial communities within groups became more similar as pigs grew. McCormack et al.  reportedLactobacillus and Barnesiella [Escherichia coli, Salmonella spp. and Clostridium spp. [Lactobacillus was high in pigs with high G:F. Lactobacillus or lactic acid bacteria makes acidic environment in the intestine and inhibit the growth of intestinal pathogens [Barnesiella also has the ability to restrict the growth of intestinal pathogens and to limit colonization of antibiotic resistant pathogens. In addition, Prevotella plays an important role in the utilization of complex sugars by biodegrading and fermenting carbohydrates in the digestive system of non-ruminants [Prevotella may impair the establishment of more effective nutrient harvesting microbiota because of the interaction between Prevotella and other beneficial microbes [Our results of the taxonomic analysis of sequence reads are consistent with the results reported by previous studies. The dominant bacterial phyla were Firmicutes and Bacteroidetes ,59,61,65nesiella ,56,66,67nesiella , which cium spp. ,70,71. Vium spp.  found thathogens . Barnesiuminants . HoweverGround brown rice can be added to growing-finishing pig diets by substituting corn up to 100% without negative effects on growth performance, nutrient digestibility, and carcass characteristics of pigs and may modify gut microbiota of pigs if it is fed for a long time.


Biomarkers which predict future health outcomes are key to the goals of precision health. Such biomarkers do not have to be involved in the causal pathway of a disease, and their performance is best assessed using statistical tests of clinical performance and evaluation of net health impact. DNA methylation is the most commonly studied epigenetic process and represents a potential biomarker of future health outcomes. We review 25 studies in non-oncological paediatric conditions where DNA methylation biomarkers of future health outcomes are assessed. Whilst a number of positive findings have been described, the body of evidence is severely limited by issues with outcome measures, tissue-specific samples, accounting for sample cell type heterogeneity, lack of appropriate statistical testing, small effect sizes, limited validation, and no assessment of net health impact. Future studies should concentrate on careful study design to overcome these issues, and integration of DNA methylation data with other ‘omic’, clinical, and environmental data to generate the most clinically useful biomarkers of paediatric disease. Precision health  can be defined as interventions “targeted to the needs of individual patients on the basis of genetic, biomarker, phenotypic, or psychosocial characteristics that distinguish a given patient from other patients with similar clinical presentations” . It is aBiomarkers that allow prediction of future health outcomes are key foci of precision health . These m3) to the fifth carbon of a cytosine nucleotide within a cytosine-phosphate-guanine dinucleotide (CpG) [Epigenetic processes regulate gene expression, without changes in the underlying DNA sequence . These ade (CpG) . CpG sitde (CpG) . CpG islde (CpG) , 13.FigAs disease outcomes are often thought to be due to a combination of genotype, environmental exposures, and the interaction of the two , DNA metThe aim of this review is to summarise the evidence regarding the use of DNA methylation as predictive biomarkers in children with a diagnosed disease. This includes prediction of disease severity, treatment response, or development of complications. Studies examining cancer have not been included as reviews in this area have been recently published –17. In aArticles were identified via the search strategy in Table SLC6A3 showed the greatest difference between preterm and term infants (increased methylation in preterm) and that increased methylation in both term and preterm infants was associated with significantly poorer motor function  and mental function . The sites of differential methylation identified in each of these studies did not overlap, which may reflect differences in sample size, the biospecimen used for methylation analysis, outcome assessment tool, and age at both methylation analysis and outcome assessment.Premature infants are known to have an increased risk of later life neurocognitive impairment. Two studies have attempted to identify DNA methylation biomarkers of future cognitive impairment in premature infants. Tilley et al.  examined4 T cells at enrolment and then 2–4 years later. Subjects were aged between 11 and 15 months at enrolment and defined as being food allergic based on a positive skin prick test and clinical reactivity. At reassessment, patients were classified as having persistent food allergy  or resolved allergy. The investigators examined whether the change in DNA methylation profiles from baseline to reassessment could predict the likelihood of persistent allergy, and they focused on 26 loci which were demonstrated to be differentially methylated between patients with and without food allergy. They found that in 24 of 26 loci, there was a significant change  in methylation from baseline to follow-up in patients with persistent allergy, whereas methylation was stable in patients whose allergy resolved. In the group with persistent allergy, the maximum change in methylation at an individual locus between baseline and follow-up was 17.2%. Fu et al. [ADRB2) in 182 children with asthma aged between 5 and 12 years. Blood was collected at enrolment, and patients were followed up for 12 months. Parent-reported symptoms were used to calculate an asthma severity score, which was used to classify patients into mild or severe disease. Multivariate logistic regression (adjusted for age and gender) was used. DNA methylation was classified as ‘low,’ ‘intermediate,’ and ‘high,’ and asthma as ‘mild’ or ‘severe.’ In a dose-dependent manner, increasing methylation of ADRB2 was associated with more severe asthma, with those with intermediate methylation having an OR 4.11 (95% CI 1.58–10.73) for severe asthma and those with high methylation having an OR 7.63 (95% CI 3.02–19.26). The median methylation level in the severe asthma group was 1.14% (range 0.2 to 3.6%) and in the mild asthma group was 0.81% (0 to 2.4%). They also examined nitrous oxide exposure and found that in the highly exposed group, those with ADRB2 hypermethylation had an OR of 4.59 (95% CI 1.03–20.55) of having severe asthma.In a large study examining 65 patients with food allergy, Martino et al.  assessedu et al.  studied VNN1) methylation in nasal epithelial cells, a surrogate for lower airway epithelium, obtained in 18 children aged 5–18 years at presentation to hospital with an acute asthma exacerbation and again after 12–24 h. VNN1 was identified as a gene of interest via an initial gene expression investigation of > 20,000 genes. Patients were treated with a standardised asthma protocol which included criteria for discharge. Good corticosteroid response was defined as a length of hospital stay ≤ 24 h, whereas poor response was defined as a length of stay > 24 h. Those who had a good response to corticosteroids had an increase in methylation at a single CpG, located in the promoter region of VNN1, whereas those that did not respond to corticosteroids had evidence of decreased methylation, although the raw figures were not reported. When assessed using Fischer’s exact test, the differences were significant (p = 0.003). At the same hospital, Zhang et al. [LDHC) at presentation was associated with poor treatment response. The mean (standard error) methylation level was 87.7% (2.1%) in the poor response group, and 78.6% (2.4%) in the good responders (p = 0.007). A major strength of the two studies looking at treatment effect was the use of nasal epithelial cells to assess DNA methylation, which is a better surrogate for bronchial epithelial cells than peripheral blood [Two studies have investigated whether DNA methylation markers can predict response to corticosteroids during an acute asthma exacerbation. Xiao et al.  studied g et al.  used theal blood .p value < 10−6). The effect size of each individual CpG was not described. The probes were located in the promoter region of associated genes (4/9), intergenic regions (3/9), and the gene body (2/9). A further 9 probes associated with intellectual disability, 3 with schizophrenia, 2 with behavioural disorders, and 8 with development disorders. The individual effect size was not detailed for any of these associations. The most common location of probes associated with intellectual disability was the promoter (4/9), and for development disorders, it was the body (4/9). Several studies have examined whether DNA methylation biomarkers can predict response to treatment of psychiatric conditions. Gasso et al. [HTR1B), measured in blood, could predict treatment response to 12 weeks of the selective serotonin reuptake inhibitor medication, fluoxetine. They studied 83 children aged between 10 and 17 years who were treated with fluoxetine due to major depressive disorder, obsessive-compulsive disorder, or generalised anxiety disorder. Subjects completed multiple validated scales which measure disease severity, and the change in these scales across 12 weeks was used to define treatment response. Pearson’s test was used to assess correlation, and a Bonferroni correction for multiple testing was performed. When the methylation level at the 7 CpG sites was averaged, they found a moderately strong inverse relationship with higher methylation level associated with reduced fluoxetine response . The methylation level for the two groups was not reported. In a study of 116 children aged 6–13 years with an anxiety disorder, the response to cognitive behaviour therapy (CBT) was assessed [SLC6A4) was performed pre- and post-treatment with CBT. Treatment response was based on whether the anxiety disorder had resolved. Subjects with a sustained CBT response (measured 6 months post-treatment) were more likely to have increased methylation at a single CpG during treatment (+ 3.48%), whereas non-responders were more likely to decrease (− 5.44%), and this relationship was significant when corrected for multiple testing (p = 0.002). In another study also examining buccal swabs from children (n = 98) with anxiety disorders, and their response to cognitive behaviour therapy, DNA methylation of CpG sites in the promoter region of FK506 binding protein 5 (FKBP5) and glucocorticoid receptor (GR) was assessed before and immediately after treatment [FKBP5 or GR methylation was found. In a study of 111 children, mean age 9.25 years, with attention deficit hyperactivity disorder (ADHD) who were treated with methylphenidate, dopamine transporter (DAT1), and dopamine receptor D4 (DRD4), promoter methylation was assessed in blood samples collected pre-treatment to see if this predicted treatment response [DAT1 methylation was associated with greater treatment response  although the mean methylation levels were not reported.Another area which has been widely studied in psychiatric disorders. In a group of 164 patients with 22q11 microdeletion syndrome, who are at an increased risk of psychiatric disorders, Starnawska et al.  performeo et al.  examinedassessed . Using breatment . The chiresponse . The diaOPRM1) promoter methylation in the saliva of 58 newborns and the relationship with NAS severity. NAS severity was primarily defined based on the need for pharmacological treatment, which was determined based on a standardised protocol. They used linear and logistic regression models as well as correction for multiple testing using the Benjamini and Hochberg method. The study found infants needing pharmacological treatment for NAS had higher methylation at several CpGs: CpG − 18 , CpG − 14 , and CpG + 23 . The same group of authors conducted another study with the same methods and research questions in a group of 86 infants with NAS. They did not replicate the finding with regard to CpGs − 18, +23, but did find that hypermethylation of CpG − 14 was associated with greater severity of NAS measured by need for 2 medications to control symptoms (adjusted difference = 4.9% (95% CI 1.8–8.1%), p = 0.0030) [ABCB1) promoter, cytochrome P450 family 2 subfamily D member 6 (CYP2D6) exon 1, and OPRM1 promoter and exon 1. They used a standardised protocol for monitoring NAS severity. Unlike the studies by Wachman et al., they did not find any significant effect. A potential explanation is the smaller sample size of 21, as compared to 58 and 86 in the positive studies.Three studies have attempted to identify DNA methylation biomarkers of severity of neonatal abstinence syndrome (NAS). Wachman et al.  analysed 0.0030) . In a st 0.0030)  also useGR promoter was analysed in blood from day 4 of life [p = 0.0486). Dhas et al. [t tests found those who developed renal failure as a complication of sepsis had slightly higher global methylation levels .In a group of 20 premature infants, DNA methylation in the  of life . The outs et al.  investigTwo studies have investigated children with inflammatory bowel disease (IBD). Howell et al.  used intU test to compare the mean methylation level and progression status. Mean methylation at cg01374129 was lower in the group that progressed , and when a receiver operator curve was calculated, the area under the curve was 0.827 (95% CI 0.780 to 0.876), with a sensitivity of 76.4% and a specificity of 85.6% at a cutoff level of 15.1%. Two studies have attempted to identify biomarkers of development of chronic pain complications post-scoliosis surgery. In a study of 128 adolescent patients with idiopathic scoliosis undergoing scoliosis surgery, OPRM1 promoter methylation was assessed at 22 CpG sites in blood collected during the operation [p < 0.05. There was no correction for multiple testing. After adjustment for preoperative pain score and morphine consumption over postoperative days 1 and 2, higher OPRM1 promoter methylation at 3 CpG sites was associated with increased acute postoperative pain. The strength of the association was weak for two CpG sites with a 1% increase in DNA methylation associated with regression coefficients of 0.921 and 1.864; however, one CpG had a regression coefficient of 17.736. When chronic postsurgical pain was assessed, there was significant different methylation at two CpG sites. The OR for these associations were relatively weak: 1.069  and 1.037 (1.000–1.075). Once again, adjustment was performed for preoperative pain and postoperative morphine consumption. The two CpG sites did not overlap with those associated with acute postoperative pain. The authors provided further evidence for the role of OPRM1 methylation via using functional genomic analysis from publicly available databases which showed increased methylation would lead to reduced gene expression and reduced response to opioids. In a subsequent study, the same authors assessed 46 adolescents undergoing scoliosis surgery. They performed an EWAS using the EpicArray on peripheral blood samples. For this study, they used medication use and questionnaires to assess pain severity. Surrogate variance analysis was used to control for confounders, and adjusted linear regression models were used to test for DMP. They identified 637 significantly differentially methylated probes associated with chronic postsurgical pain (p < 0.05). The DMPs were most commonly found in the promoter region (23.31%) and gene bodies (36.34%). The 637 DMPs were associated with 310 genes and which are associated with pathways such as GABA receptor signalling, protein kinase C signalling, dopamine receptor, and cyclic adenosine monophosphate-mediated signalling.Meng et al. studied patients with idiopathic scoliosis and the risk of progression of the scoliosis curve beyond 30°, which is a marker of severity. They identified CpG sites of interest in blood, by first investigating two female twin pairs with idiopathic scoliosis who were discordant for curve progression. The top 4 DMP were chosen for replication in a cross-sectional cohort of 92 adolescents who were divided based on the severity of scoliosis at presentation, and only one CpG site (cg01374129) remained a significant DMP. This CpG site is not associated with a gene. They then tested the performance of methylation at this individual CpG in a cohort of 276 adolescent subjects who had mild scoliosis at presentation and then were divided based on whether their scoliosis progressed prior to skeletal maturation. They used the Mann-Whitney peration . Acute a4 T cells collected as anti-tumour necrosis alpha treatment was stopped. Weighted gene coexpression network analysis was used to identify differential methylation. This analysis examines correlated CpGs rather than individual CpGs and reports differentially methylated regions (DMR). They identified 5 DMRs between the two groups. The DMRs were associated with genes implicated in T cell activation, and this relationship was confirmed in subsequent gene expression and protein analysis experiments, with elevated markers of T cell activation in those who did not have sustained remission post-therapy cessation. Kuo et al. [FCGR2A) promoter methylation and response to treatment with intravenous immunoglobulin (IVIG), based on defervescence within 48 h. The sample source for methylation analysis was not reported. Mean methylation levels between responders and non-responders were compared using the chi-square test. They identified 5 CpG sites where increased methylation was associated with non-response to IVIG. At the 5 CpG sites, the difference in mean methylation level between the two groups ranged from 9.75 to 18.94%, and at all sites, the relationship was strong . Yang et al. [t test. No relationship between mean DNA global methylation level and ACTH treatment effect was identified. In a group of 97 patients with Beckwith-Wiedemann syndrome, Gaston et al. [KCNQ1OT and H19 methylation and risk of tumour development (a known complication of Beckwith-Wiedemann syndrome). They used blood samples, and in some patients, tissue samples from affected organs were also obtained. Using the Kaplan-Meier method, abnormal H19 methylation (defined as a methylation index > 60%) was strongly associated with an increased risk of tumour development .A number of other conditions have been investigated by single studies. A single study has investigated 68 children with juvenile idiopathic arthritis . Patiento et al.  studied g et al.  studied n et al.  examinedSeveral studies have investigated the use of DNA methylation as a biomarker to predict future health outcomes in children. In support of the notion that DNA methylation is a viable biomarker, 22 of the 25 studies report a positive finding, although that may represent publication bias . UnfortuOne of the most important aspects of any biomarker study is to ensure that the outcome of the study is robust and clinically relevant. A number of studies have used validated and widely accepted tools for assessing the outcome, such as the use of the Bayleys Scale of Infant Development for assessing neurocognitive development. Where possible, gold standard outcome measures should be used as they are more robust than other outcome measures such as parent-reported asthma symptoms, which are open to error . This isAnother important consideration is the tissue source for DNA methylation analysis. As a large proportion of methylation variation is tissue-specific , it is dWhere samples used for DNA methylation analysis involve multiple cell types , it is important that cell composition of the sample is accounted for in the analysis . As eachThe statistical analyses used to date limit the clinical applicability of the evidence that has been generated. For a test to be used in clinical practice, the performance should be assessed via statistical measures such as sensitivity, specificity, positive predictive value, negative predictive value, and area under a receiver operator curve. Only one  of the iOPRM1 methylation in NAS, with two studies finding a significant association [A major limitation to the evidence generated in this field so far is the generally reported small disease-associated effect sizes of differential methylation between groups. These likely arise from a combination of the limited sensitivity and specificity of current methods used to assess DNA methylation, relative to the gold standard whole-genome bisulphite sequencing  in combiociation  and one ociation . A numbeDespite the limitations of the evidence so far, there is still potential for useful DNA methylation biomarkers to be identified. Whilst most of the issues raised can be overcome by careful study design, the small observed effect sizes may be more difficult. One approach would be to incorporate DNA methylation data into combined predictive models. These models could pool data from multiple ‘omic’ inputs such as the genome (genetic mutations), methylome (DNA methylation), transcriptome (gene expression), and proteome (protein levels). An example of a program which is using the proposed multi-omic approach to better understand disease pathology and predict disease outcomes is the Integrative Human Microbiome Project. The second phase of this project is collecting biospecimens from patients to investigate the onset of inflammatory bowel disease and type 2 diabetes and is analysing host genome, methylome, transcriptome, and proteome as well as other factors such as the microbiome . The omiIf the goals of precision health are to be recognised, biomarkers which predict future health outcomes will need to be identified. With the expansion in ‘omics’ technology, there are multiple tools which may aid discovery of such biomarkers. One such tool is epigenetics, in particular DNA methylation, and multiple studies have already undertaken research in this area. Whilst several positive findings have been made, there is a long way to go before these findings can be incorporated into clinical care. To ensure the validity of any future discoveries, researchers should ensure consideration is given to disease outcomes, tissue-specific specimens, adjustment for cell-type heterogeneity, appropriate statistical tests, replication of positive findings, and incorporating DNA methylation data into combined biomarkers. If these factors are considered, DNA methylation biomarkers have the potential to improve the healthcare delivered to children.Additional file 1: Table 1. Included articles.


The rats were anaesthetized with diethyl ether, and blood samples were obtained for sex hormonal analysis; caudal epididymis was dissected for sperm count, motility, and viability, and the testis were homogenized for lipid peroxidation and proton pump  activity. Proton pump activity was assayed spectrophotometrically using the Stewart method to determine the inorganic phosphate level. Histopathological changes of the testis were also studied. The group treated with CdCl2 showed a significant (p < 0.05) decrease in proton pump activity, sperm count, and motility and a significant (p < 0.05) increase in malondialdehyde level when compared with the control group. The CdCl2-treated group also showed decrease reproductive organ weights and hormonal levels and cause necrosis of spermatogonia lining the seminiferous tubules. Rats treated with vitamin E orally for 30 days prior to CdCl2 exposure showed improvement in proton pump activity, a significant (p < 0.05) increase in sperm parameters and luteinizing hormonal level, and a decrease in the lipid peroxidation level as compared with the CdCl2 group. This study showed that vitamin E ameliorated the toxic effect of CdCl2 on proton pump activity in the testes, hence improving testicular integrity, structures, and functions.Proton pumps are membrane-bound enzymes important in generating gradients that help in maintaining cellular ion homeostasis, cell membrane potential, water, and solute transport across the cell surface. This study investigated the modulatory role of vitamin E on proton pump activity and reproductive parameters in cadmium-induced testicular damage. Twenty (20) male Wistar rats weighing between 180 and 200 g were sorted into 4 groups of five rats each. Group I served as the control and was given normal saline orally, Group II rats were treated with a single dose of 2 mg/kg BW cadmium chloride (CdCl Vitamin E is synthesized only by plants and, therefore, is found in plant products; the richest sources include vegetable oils and to a lesser extent, seeds, nuts, and cereal grains. It is particularly effective in preventing lipid peroxidation, a series of chemical reactions involving the oxidative deterioration of polyunsaturated fatty acids (PUFAs) [Infertility is defined as the lack of ability of a noncontracepting couple to conceive after repeated and unprotected sexual intercourse for more than one year . The mal sources . Cadmium sources . Exposur sources , 8. Expo sources . Testicu sources . Exposur sources  and a de sources . Cadmiumtaken in . In epitrmatozoa  and in m (PUFAs) . Vitamin (PUFAs) –17. VitaCadmium chloride  and vitamin E  were purchased from their local representatives in Nigeria. All other chemicals and reagents used in this study were of analytical grade.ad libitum throughout the period of the experiment. The use and care of the animals and the experimental protocol were in strict compliance with the Institute of Laboratory Animals Research (ILAR) guidelines, 1996 [Twenty (20) adult male Wistar rats weighing between 180 and 200 g obtained from the National Veterinary Research Institute, Vom, Jos, Nigeria, were used in this study. The animals were brought to the Department of Physiology, Bingham University animal house facility, where they were acclimatized for two weeks before the start of the experiment. They received food (grower's feed) and water The animals were randomly divided into four groups of five rats each as follows:Group I: animals in this group were given normal saline orally for 30 days and served as controls2 (2 mg/kg BW) intraperitoneally on day 30 of the experimental periodGroup II: animals in this group were treated with a single dose of CdClGroup III: animals in this group were given vitamin E (100 mg/kg BW) orally for 30 days2(2 mg/kg BW) intraperitoneallyGroup IV: animals in this group were pretreated with vitamin E (100 mg/kg BW) for 30 days orally and then administered with a single dose of CdClIn all groups, blood samples were collected retroorbitally; sera obtained were stored at 4°C for subsequent ELISA analysis for the determination of reproductive hormones. The rats were anaesthetized with diethyl ether, and the testes were collected, cleared of adherent tissue, and weighed using a sensitive weighing balance.The left epididymis of each rat was used for the determination of epididymal sperm concentration using the Neubauer hemocytometer, while % sperm motility was determined as previously described by Sonmez et al. . The epiMalondialdehyde levels were estimated by the method previously described by Evans . This waThe activities of sodium/potassium, calcium, and magnesium ATPase enzymes were assayed by the modified method previously described by Evans et al.  and Stewp < 0.05.Data are presented as mean ± SEM. One-way ANOVA was performed with the Statistical Package for the Social Sciences (SPSS package version 16) to test the significance of differences between the results. The difference was considered significant at p < 0.05). Treatment with vitamin E resulted in a significant weight gain by 46 g after 30 days at levels approaching the control group, and treatment with vitamin E for 30 days orally and then administration with a single dose of cadmium chloride (2 mg/kg BW) resulted in weight gain (25.8 g) at levels approaching the control group (Groups II and III versus Groups I and IV). Mean testicular weight in the cadmium chloride group significantly (p < 0.05) decreased as compared to that of the control group, while treatment with vitamin E for 30 days orally and then administration with a single dose of cadmium chloride (2 mg/kg BW) decreased compared to treatment with vitamin E for 30 days orally increased significantly (p < 0.05).The control group (I) gained weight over the thirty days of the experimental period, with the mean body weight increasing by 32.4 g after 30days . In contp < 0.05) decrease in the lipid peroxidation level compared to the cadmium chloride group while the group treated with cadmium chloride showed a significant (p < 0.05) increase in lipid peroxidation level compared to the control group. Treatment with vitamin E orally for 30days prior to cadmium chloride administration intraperitoneally showed a significant (p < 0.05) increase in proton pump activity, sperm count, and motile sperm cells and a decrease in nonmotile sperm cells when compared with the cadmium chloride group  increase in serum LH level and a significant (p < 0.05) decrease in FSH and testosterone levels. Inhibition of serum levels of LH, FSH, and testosterone by cadmium chloride resulted in altered spermatogenesis evident by a decrease in sperm count shown by the cadmium chloride group (The cadmium chloride group  showed ade group . This abThe testis of rats in the control group  exerted p < 0.05) decrease in lipid peroxidation level. Studies have shown that within two weeks, cadmium chloride would have affected the detrimental effect on spermatogenesis; thus, the treatment of vitamin E for 30 days for the antioxidant protection [+-coupled secondary transport of H+, Ca2+, glucose, amino acids, and neurotransmitters across the plasma membrane [+/K+ ATPase function by modification at specific sites in a selective manner [In this study, the cadmium chloride group  showed aotection , 26. Thiotection . Testicuotection . Membranmembrane . Lipid pe manner .p < 0.05) decrease in proton pump activity in the cadmium chloride-treated group as compared with control. ATPases  are very sensitive to the presence of metal ions such as cadmium and lead [2+) competes with ATPase cofactors  and form complexes with its functional group by nonspecific binding with sulfhydryl groups of cysteine residues leading to the loss of protein-SH groups, which is a consequence of lipid peroxidative damage [+/K+ ATPase helps create the appropriate medium in the seminiferous tubule lumen for normal development of the spermatozoa [p < 0.05) in sperm count associated with the cadmium-treated group when compared with the vitamin E and control groups. This is because the transmembrane distribution of electrical charges, resulting from the uneven Na+ and K+ transport activity of the Na+/K+-ATPase, contributes to maintain the resting membrane potential that provides cells with the excitability required for their movement, contraction of myosin and actin filaments on the peritubular cells of the seminiferous tubule, and transmission of impulses [p < 0.05) increase in proton pump activity, sperm count, an dmotile sperm cells and a decrease in nonmotile sperm cells when compared with cadmium-treated group  in the testis. Vitamin E in the diet is encouraged as it is effective in neutralizing reactive oxygen species generated from cadmium and modulating the activity of the membrane-bound enzymes necessary for the maintenance of testicular function and integrity. This will therefore contribute to improving the fertility of among males who are exposed to heavy metals.This study showed that vitamin E ameliorated the toxic effect of cadmium on the proton pump activity (Na


Genome-wide methylation analysis was conducted using Infinium Human Methylation 450 BeadChip  of q < 0.05 was applied). Expression levels were measured using HumanHT-12v4 Expression BeadChip . The integrative analysis of both array data identified four genes  with higher methylation and lower expression level in pre-surgery women compared to normal weight women: and two genes (i.e. ZFP36L1 and USP32) that were differentially methylated after BS. These methylation changes were in promoter region and gene body. All genes are related to MAPK cascade, NIK/NF-kappaB signaling, cellular response to insulin stimulus, proteolysis and others. Integrating analysis of DNA methylation and gene expression evidenced that there is a set of genes relevant to obesity that changed after BS. A gene ontology analysis showed that these genes were enriched in biological functions related to adipogenesis, orexigenic, oxidative stress and insulin metabolism pathways. Also, our results suggest that although methylation plays a role in gene silencing, the majority of effects were not correlated.DNA methylation could provide a link between environmental, genetic factors and weight control and can modify gene expression pattern. This study aimed to identify genes, which are differentially expressed and methylated depending on adiposity state by evaluating normal weight women and obese women before and after bariatric surgery (BS). We enrolled 24 normal weight (BMI: 22.5 ± 1.6 kg/m Methylation patterns are established in early life and can be remodeled in adult cells, by modulating DNA interactions with proteins and transcription factors3, being able to alter gene expression profile4.DNA methylation in CpG dinucleotides is a dynamic process and best characterized epigenetic modifications5. In line of this, epigenetic changes may predispose a disease risk or can occur once a disease has developed6. Thus, DNA methylation provides a link between environmental, genetic factors and weight control.Weight gain or loss during infancy and adulthood, by changing the energy storage and adipose tissue homeostasis, may alter molecular mechanisms and biological processes7, being associated to a public health burden. In the context of obesity treatment, bariatric surgery (BS) has been the best choice for cases of severe obesity and has been shown to be the most effective way to promote significant and sustained weight loss8. Among the surgical techniques, Roux-en-Y gastric bypass (RYGB) is the most performed in Brazil and worldwide, and is considered a gold standard because its high efficacy and low morbid-mortality9. However, inter-individual variability of the response to bariatric surgery, mainly related to weight loss, is evident10, and the unpredictability of weight-loss success represents a significant barrier in the surgical management of obesity11.Obesity has reached epidemic proportions and, in 2016, affected about 1.9 billion adults worldwide13. In addition, the role of epigenetic signature in metabolic results after obesity treatment has been described previously15. Knowing that nutrition is one of the greatest environmental stimuli that can change gene methylation/expression signature16, some studies have demonstrated that BS changes DNA methylation levels17. Other publications have evidenced that weight loss by surgery is also able to modify gene expression levels, mainly of genes related to metabolic pathways19.Recent epigenome-wide association studies (EWAS) evidenced multiple DNA methylation loci associated with body mass index (BMI)21; however, to gain a broader perspective about the interface between CpG methylation and functional effects in transcription, we performed an integrated analysis with methylation and expression data. Thus, the objective of the current study was to identify genes, which are differentially expressed and methylated depending on adiposity state by evaluating normal weight women and obese women before and after BS.The relevance of differential DNA methylation and gene expression consequent of BS was previously evidenced by our research groupp < 0.001), BMI  and fat mass  reduction.No significant difference was found between the ages of each group . Normal weight women showed a mean weight of 60.7 ± 6.3 kg and mean fat mass of 29.6 ± 4.1%. After 6 months of BS, we evidenced a significant weight  were differentially methylated between obese women before BS and normal weight women, and most of them showed high methylation level in obese patients. Also, 666 CpGs (located in 495 genes) had methylation levels changed after RYGB. For the microarray gene expression analysis, a total of 600 genes were differently expressed (DEGs) between obese women before BS and normal weight patients. Indeed, 1,366 genes were differentially expressed between surgery times. Among these, 88 genes were up- and 512 genes were down-regulated.p < 0.05) and differently methylated CpG sites . When preoperative patients and normal weight women were compared, a total of six genes were, concomitantly, differentially methylated and expressed  present their methylation and expression levels in the same direction: both lower in preoperative patients compared to normal weight women. However, four genes  presented methylation and expression levels in opposite direction: higher methylation and lower expression level in pre-surgery women compared to normal weight women. Also, these CpG sites were located at TSS200, TSS1500 and 5′UTR regions (gene promoter region).Figure sed Fig. . Two of ZFP36L1 and USP32) were differentially methylated and expressed after RYGB. Methylation and expression levels of ZFP36L1 and USP32 genes were changed in opposite direction. For ZFP36L1, the methylation level decreased after surgery while its expression increased. In contrast, for USP32, methylation level increased and expression decreased after the surgery. Considering CpGs location, we observed that they were located in gene body and TSS1500 region.Considering pre and postoperative times comparison, Venn diagram analysis identified a total of eight genes that were common in methylation and expression arrays Fig. . Of thesThe expression analysis between postoperative and normal weight individuals showed no differential gene expression, thus concomitant analysis between methylation and expression profile couldn’t be performed.Table To additional understanding about the biological relevance of the identified genes, GO analyses were performed. These analyses showed that these genes are related to MAPK cascade, NIK/NF-kappaB signaling, cellular response to insulin stimulus, proteolysis and others.We identified in the present study genes that are concomitant differentially methylated and expressed in leukocytes after BS. Relevantly, these genes were involved in molecular pathways related to obesity´s physiopathology. Also, comparing obese subjects (before BS) and normal weight women, we identified four genes that showed lower expression and higher methylation levels; which were related to adipogenesis, anti-satiety effect, oxidative stress, and insulin metabolism. Moreover, we evidenced that two genes related to proteolysis and adipogenesis, were epigenetically regulated by RYGB procedure.23, results obtained here showed that promoter methylation (TSS200 and TSS1500) is associated with decreased expression; however, for specific CpG sites, intragenic methylation (gene body) also correlates with decreased gene expression. Grundberg et al.24, found that a large number of methylation and expression associations were positive, thus the increase in methylation levels was linked to an increase of corresponding gene expression. More interestingly, evidence suggested there an adjustment mechanism of body gene methylation levels for transcription regulation. In line of this, when promoter methylation is constant, increasing body methylation is associated with more repressed expression25. Thus, considering that some authors affirmed that the function of DNA methylation in intergenic and gene-body regions is less defined24, our results showed that regardless of the location of methylated cytosine (promoter region or body), DNA methylation promotes a reduction in gene expression.Despite the fact that the impact of DNA methylation on gene expression seems to depend of cytosine methylated site and locationCCNL1 and SLC5A8) and six genes  were not regulated by DNA methylation in obese versus normal weight and preoperative versus postoperative comparisons, respectively. Study with cancer cells showed that the presence of methylation does not always imply in gene silencing26. Thus, our results showed that despite methylation plays a role in gene silencing, the majority of effects were not correlated.In addition, it is important to highlight that the expression of two  and adipogenesis stimulus, pointing to an important role of TPP2 in obesity28. Thus, TPP2 seems to have dual role in metabolic homoeostasis, affecting both feeding behavior and adipose tissue biology per se28. Moreover, TPP2 is also related to different isoforms of protein convertase gene family (PSCK) that are associated with obesity related traits (e.g PCSK1 and PCSK2)29.Another point to be discussed is that the present study is the first to identify an association between ARL6IP1 and PSMC6 have been associated with insulin synthesis and pathology of diabetes, respectively31. Also, ARL6IP1 protein is associated phosphatidylethanolamine-binding family of proteins (PEBP1), which has important role in MAPK, NF-kappa B, and glycogen synthase kinase-3 (GSK-3) signaling pathways. PSMC6 gene encodes one of the ATPase subunits and its expression was inversely correlated with BMI11.On the other hand, FAM49B has been appointed as novel regulator of mitochondrial function and may thus be associated with oxidative stress and inflammation, however, up to now, no functional data about this protein has been published32. Furthermore, USP32 is a highly conserved gene and uncharacterized gene that its stable silencing caused a significant decrease in the proliferation and migration rate of cells9. For this reason, USP32 has been associated with growth rate of cancer cells33.Lastly, ZFP36L1 and USP32 methylation and expression levels in leukocytes. In culture 3T3-L1 preadipocytes, ZFP36L1 was associated with adipogenesis34 by regulating adipogenesis rate, playing an important role in obesity development35. According to Tseng36, ZFP36L1 overexpression might repress adipogenesis at least by down-regulating PPARG2 expression. These findings are a proof that blood leukocytes are able to reflect the regulation of same adipose tissue biology-related genes as it was also demonstrated in previous reports39. In the current research, we were unable to detect differences in methylation levels of previously identified obesity related CpG sites, probably because differences in the study design, sample size or the threshold used for selection of candidates. However, the current study adds new information to this issue. In fact, the identified genes were involved in adipose tissue-related pathways that were also observed in previous reports on obesity-related methylation profile39.We also identified that bariatric surgery modify The use of DNA and RNA extracted from leukocytes is a limitation of this study because both, expression and methylation, are tissue specific molecular mechanisms. However, leukocytes samples are less invasive, more convenient and acceptable than biopsies of target tissues (i.e adipose tissue) to perform longitudinal assessments and more suitable in clinical practice. To search for obesity-associated epigenetic biomarkers for the diagnosis and management of the disease is a huge challenge as adipose tissue is inaccessible without surgery. As mentioned above, in the obesity field, relevant studies have been recently published providing evidence that blood cells can be used to identify robust and biologically relevant epigenetic variation related to BMI. Moreover, it was demonstrated that epigenetic biomarkers in blood could mirror age-related epigenetic signatures in biologically relevant target tissues such as adipose tissue. Therefore, blood leukocytes are suitable, minimally invasive biological sources to evaluate obesity signatures. On the other hand, even though the identified changes in methylation and expression pattern induced by bariatric surgery in the present study could be transient, long-term studies would indicate the degree of plasticity of the epigenomic changes associated with bariatric surgery and would establish if over a longer time, the methylation profiles returned to the obese-pre surgery levels.In conclusion, integrating analysis of DNA methylation and gene expression evidenced that there is a set of genes relevant to obesity that changed after BS. A gene ontology analysis showed that these genes were enriched in biological functions related to adipogenesis, orexigenic, oxidative stress and insulin metabolism pathways. Also, our results suggest that although methylation plays a role in gene silencing, the majority of effects were not correlated.2, 36.9 ± 11.8 years) and 24 severe obese women  submitted to RYGB which methylation and gene expression profile had been analyzed previously21. All samples from obese women were collected at preoperative time and after 6 months of bariatric surgery. Normal weight women were evaluated once.The present study included 24 normal weight women  and an anastomosis of the gastric stump to the jejunum (both remaining loops measured about 100 cm). The surgical procedure was standardized at our hospital following the most usual pattern performed worldwide and well describedThe study was conducted with the approval of the Hospital Ethics Committee and in agreement with the Declaration of Helsinki. Informed consent was obtained from all individual participants included in the study. All methods were carried out in accordance with relevant guidelines and regulations.21, genomic DNA was extracted from peripheral mononuclear blood cells (PMBC) using GE Health Care kit (Illustra blood genomic Prep Mini Spin kit) and this extracted DNA was stored at −80 °C until the next steps. DNA fragmentation or RNA contamination was analyzed by 1% agarose gel electrophoresis. Also, DNA was bisulfite converted using EZ DNA methylation kit Methylation-Gold  according to the manufacturer’s instructions and then immediately hybridized in BeadChip. Genome-wide methylation analysis was conducted using the Infinium Human Methylation 450 BeadChip . Beadchips were scanned with the Illumina iScanSQ system and DNA quality checks, bisulfite modification, hybridization, data normalization, and statistical filter were performed as described before21.As aforementionedAnalysis of methylation data were conducted using the Genome Studio software version 2011.1 (Illumina Inc.) and methylation levels were expressed in beta values (β). β values that were calculated as the intensity of the methylated channel divided by the total intensity / + Max  + 100), and ranged from 0 (unmethylated) to 1 (fully methylated). β values with detection p-values > 0.01 were removed from analysis because were considered to fall below the minimum intensity and threshold. Also, probes that were localized to the sex chromosomes and those CpGs that contained single nucleotide polymorphisms were filtered out.p were adjusted for multiple comparisons by using the false discovery rate . Also, a threshold for the significant CpG sites based on Δβ with a minimum value of 5%  and FDR < 0.05 was applied. We used R software (version 3.2.0) to perform these analyzes.Differences in methylation levels between preoperative and postoperative groups (Δβ) was calculated for a given CpG site by subtracting the mean beta value from the pool of pre-surgery samples (pre-surgery period) as compared to the pool of samples collected after six months of RYGB and were tested by t test. Values of 41. Extracted RNA was stored at −80 °C until the next steps. After, RNA integrity number (RIN) was analyzed using Bioanalyzer . Expression levels were measured using the HumanHT-12 v4 Expression BeadChip (Illumina Inc.) Expression data were visualized and analyzed using Genome Studio Software (Illumina®). Beadchips were also scanned with the Illumina HiScanSQ system and Genome Studio Software (Illumina®) was also used. For quality control and normalization, the samples with less than 6000 significantly detected probes  were excluded and differentially expressed transcripts were identified with 95% confidence of no more than 1% false positive using an Mann-Whitney test. A cutoff of p ≤ 0.05 was used to detect differentially expressed probes. In addition, a cutoff of fold change ≥2.0  was used. p values obtained from permutations and fold change cutoff values were then used to minimize the chances of false positives. Gene expression microarray results were validated using real-time polymerase chain reaction 20.Total RNA was extracted and purified from whole blood using phenol-chloroform extraction method modified by Chomczynski & SacchiFor both methylation and expression analysis, samples from obese and normal weight women were randomly scattered on each Beadchip and not one batch was one and the other was opposite.21 were overlapped with the lists from the methylation analysis20 using Venn diagram (http://bioinfogp.cnb.csic.es/tools/venny/). Figure Before comparing gene expression and methylation analysis, a correction by cellular type was made in DNA methylation data  and KEGG signaling pathway analysis were used. The identification numbers (IDs) were loaded and analyzed against the human reference genome by means of a Bonferroni multiple test adaptation threshold of p < 0.05.Overrepresented pathways were then obtained from this new list of common genes. Genes were classified according to gene function. For this, the WebGestalt program (Gene SeT AnaLysis based on the WEB, This study has been approved by the Ethical Committee of Clinical Hospital of Ribeirao Preto School of Medicine, University of São Paulo. All patients gave their written consent for participation in the study.Supplementary Table 1.


We demonstrate the fabrication of free-standing inverse opals with gradient pores via a combination of electrophoresis and electroplating techniques. Our processing scheme starts with the preparation of multilayer colloidal crystals by conducting sequential electrophoresis with polystyrene (PS) microspheres in different sizes . The critical factors affecting the stacking of individual colloidal crystals are discussed and relevant electrophoresis parameters are identified so the larger PS microspheres are assembled successively atop of smaller ones in an orderly manner. In total, we construct multilayer colloidal crystals with vertical stacking of microspheres in 300/600, 300/1000, and 300/600/1000 nm sequences. The inverse opals with gradient pores are produced by galvanostatic plating of Ni, followed by the selective removal of colloidal template. Images from scanning electron microscopy exhibit ideal multilayer close-packed structures with well-defined boundaries among different layers. Results from porometer analysis reveal the size of bottlenecks consistent with those of interconnected pore channels from inverse opals of smallest PS microspheres. Mechanical properties determined by nanoindentation tests indicate significant improvements for multilayer inverse opals as compared to those of conventional single-layer inverse opals.  The synthesis of materials with tailored porosity and pore size has attracted considerable attention for their promising potentials in electrocatalysis, sensing, scaffolds, and capacitors ,3,4,5,6.The fabrication of inverse opals starts with the construction of colloidal crystals serving as the template to allow the filling of selective materials into the interstitial voids among the close-packed microspheres, followed by the removal of the colloidal template. Therefore, the formation of ideal colloidal crystals is critical to ensure inverse opals with desired microstructures. Conventional approaches to prepare colloidal crystals entails the packing of monodispersive microspheres via spontaneous assembly routes including sedimentation, solvent evaporation, interface control, and spin coating ,14,15,16In contrast, electrophoresis is a known process to deposit oxide polymers and small molecules for coating purposes, and many industrial applications have been demonstrated ,24,25,26In this work, we used a sequential electrophoresis approach to prepare colloidal crystals consisting of microspheres in different sizes , and used them as the template to fabricate inverse opals with gradient pores. We conducted extensive experiments to validate the critical packing rules for the successive assembly of microspheres in a vertical layer-by-layer arrangement. Because of multilayer colloidal templates without defects, the resulting multilayer inverse opals were easily removed from the substrates, forming free-standing inverse opaline membranes. In nanoindentation tests, these multilayer inverse opals revealed improved mechanical properties compared with their single-layer counterparts. 2 at 65 °C for 12 h in a thermostat reactor. Subsequently, styrene , sodium 4-styrenesulfonate , and potassium bicarbonate  were dissolved, and they were used as the monomers, comonomers, and buffer, respectively. The mixture underwent constant stirring for 15 min to form a homogeneous solution. Next, potassium persulfate  was added to initiate the polymerization step, and the polymerization lasted 16 h at 65 °C. Afterward, the mixture was cooled at 60 °C to evaporate residual solvent to retrieve PS microspheres in dry powders. In this study, we synthesized PS microspheres in diameter of 300, 600, and 1000 nm, and their respective synthetic parameters are listed in The polystyrene (PS) microspheres in various sizes were used to construct the multilayer colloidal crystals and they were synthesized by a soap-free emulsion polymerization process. In general, 1000 mL DI water was deoxygenated by N3(aq) and 1 M NH4OH(aq). To construct multilayer colloidal crystals, a sequential vertical electrophoresis technique was adopted in which a 4 × 4 cm2 Indium tin oxide (ITO) and a 10 × 10 cm2 stainless steel were used as the working and counter electrode, respectively. The electrophoretic cell contained 150 mL PS suspension and the distance between the electrodes was 1 cm. The sequential electrophoresis was conducted at 10 °C in separate PS suspensions with different PS microspheres, and the imposed electric field was adjusted accordingly. In total, we prepared multilayer colloidal crystals consisting of microspheres stacked in sequences of 300/600, 300/1000, and 300/600/1000 nm (from bottom to top). Afterwards, the multilayer colloidal crystals were retrieved and rinsed by DI water, followed by a mild heat treatment at 90 °C in air for 48–96 h. Prior to the electrophoresis process, the PS microspheres were suspended in anhydrous ethanol, and the suspension pH was adjusted by minute amount of 1 M HNO4·6H2O , 0.125 M NiCl2·6H2O , and 0.3 M H3BO3  [2 and the counter electrode was a 6 × 6 cm2 Ni plate. The electroplating was carried out in a galvanostatic mode of 2.5 mA cm−2 for 90–210 min at 25 °C. Afterward, the sample was rinsed with DI water and dried in an oven in air at 50 °C. Next, the sample was immersed in toluene  at 60 °C for 48 h, followed by a heat treatment at 350 °C for 2.5 h in 95% Ar + 5% H2 to remove the PS microspheres completely. In this study, we also prepared conventional single-layer colloidal crystals and their inverse opals for comparison purposes. To fabricate inverse opals with gradient pores, we conducted the Ni electrodeposition using the Watt’s plating formula which contained 0.5 M NiSO, SHOWA) . The wor3(aq) and NH4OH(aq). The microstructures and morphologies of multilayer colloidal crystals and inverse opals with gradient pores were observed using a scanning electron microscope . The critical pore sizes (bottlenecks) within the inverse opals were determined by a capillary flow porometer . For porometer analysis, the sample was detached from the ITO substrate as a free-standing inverse opaline film and cut to a circular shape of 1 cm diameter. The mechanical properties of inverse opals with gradient pores were determined by nano-indentation using a Bruker’s Hysitron TI 980 TriboIndenter with a Berkovich diamond indenter tip. The load was steadily increased to the maximum load of 10 mN and decreased to 0 mN at rate of 1 mN s−1. The penetration depth was kept below one-fifth of the corresponding sample thickness to minimize any substrate interference. At least 10 measurements were conducted for each sample and the resulting parameters, including hardness and reduced elastic modulus, were reported and discussed. The PS microsphere in different sizes were subjected to zeta potential measurements  to obtain their respective zeta potential. The pH values for PS colloidal suspensions were adjusted by the addition of minute amounts of 0.1 M HNOWe recognized that the key to obtain desirable colloidal crystals is to synthesize monodispersive microspheres with appreciable surface charges. Unfortunately, the size distribution for commercially available PS microspheres is rather broad (ca. 10% standard deviation) and the magnitude of their surface charges vary widely, making it extremely difficult to construct large-area colloidal crystals with reduced crystallographic defects. Therefore, in our laboratory, we have developed the necessary synthetic steps to produce uniform PS microspheres with sufficient surface charges. In addition, our processing conditions could be fine-tuned to produce PS microspheres in different diameters. 2S2O8 and NaSS from the synthesis step that produces SO42− and HSO3− ions re-adsorbing onto the PS microspheres. In an acidic solution, the PS microspheres are able to attract additional protons, rendering less negative surface charges. In contrast, in an alkaline solution, the adsorption of hydroxyl ions results in more negative surface charges. Interestingly, at identical pH, the 1000 nm microspheres revealed a consistently smaller negative zeta potential than those of 300 and 600 nm microspheres. This pattern was possibly due to its relatively smaller surface-to-volume ratio. In general, a zeta potential of ±40 mV is necessary for colloids to form a stable suspension, and a zeta potential greater than ±60 mV ensures a suspension with long-term stability. Therefore, our PS microspheres were able to form stable suspension for electrophoresis in the pH range of 2–12. Successful implementation of electrophoresis relies on a stable colloidal suspension so the zeta potential for the suspending colloids becomes critical. Electrophoresis entails the migration of charged microspheres that are driven toward a substrate with opposite polarity. According to Besra and Liu, the deposition rate for electrophoresis is linearly proportional to the suspension concentration, electric field, and zeta potential . In our Multilayer colloidal crystals require PS microspheres in different sizes so their exact packing order matters. Our experiences indicate that larger microspheres could be deposited atop of smaller microspheres provided the latter ones are arranged with a uniform surface for larger microspheres to assemble on. Otherwise, if the microspheres are stacked in opposite way, the smaller microspheres during electrophoresis might be forced to squeeze into the interstitial voids and grain boundaries of the large microspheres underneath. As a result, the smaller microspheres are more likely to be randomly-packed at the interface, which severely impacts the crystallinity of top-layer colloidal crystals. The pH of the suspension affects both zeta potential and mobility of PS microspheres in the suspension, as well as the lateral movements of microspheres on the substrate. Our experiences suggest that the first electrophoresis needs to be performed in suspension with slightly higher pH whereas the second electrophoresis requires a suspension with smaller pH. This is to ensure that the microspheres in the first-layer colloidal crystals are properly fixated in their respective positions to provide a stable interface for large microspheres to assemble on during the formation of second-layer colloidal crystals. In electrophoresis, a stronger electric field leads to a faster deposition. In order to maintain the structural integrity of first-layer colloidal crystals, the second electrophoresis is carried out under a relatively stronger electric field. It is because a greater electric field helps in ‘gluing’ the microspheres in the first-layer colloidal crystals on the substrate. However, it is noted that the effect of increased electric field is opposite to that of lower pH of the suspension for second-layer colloidal crystals. Therefore, the exact magnitude of the electric field is carefully balanced during the second electrophoresis stage to minimize physical impacts of arriving microspheres on the crystallinity of first-layer colloidal crystals. The above discussion focuses on the movement or mobility of individual PS microspheres and their effects on the crystallinity of colloidal crystals. In practice, the electrophoresis entails aggregated movements of many microspheres. Therefore, for the formation of multilayer colloidal crystals, the solid loadings for different PS suspensions need to be in a close range.2+/Ni. We recognized that the reduction of Ni2+ ions was straightforward so its charge transfer resistance was expected to be constant. Therefore, the observed reduction in voltage was associated with the decrease in electrolyte iR loss during the plating process. It is because, at the beginning, the reduction of Ni2+ took place at the bottom of colloidal crystals which represented the longest percolation length. As the Ni was filled up as time progressed, the electrolyte percolation distance became shorter, resulting in a smaller voltage. In short, from Despite the multilayer colloidal crystals being constructed with PS microspheres in different sizes, their close-packed structures in principle inferred a constant packing density of 74%, leaving 26% as the interstitial voids to be filled. Consequently, multilayer colloidal crystals consisted of different microspheres were expected to exhibit similar plating behaviors. The multilayer inverse opals demonstrated impressive structural robustness which enabled them to be detached easily from the ITO substrate, becoming essentially free-standing inverse opals with gradient pores. Therefore, it is feasible to determine the bottlenecks and their distributions using a porometer. In our samples, the bottlenecks were expected to be the diameter of interconnected pore channels residing within the inverse opals of smaller macropores, i.e., the bottom layer of multilayer inverse opals. For the sample of 300/600 nm, as shown in In the literature, it has been established that the mechanical properties of nanostructured materials are affected by their pore sizes and spatial distribution ,40,41. TH) and reduced elastic modulus (rE) were derived using following equations [A is the projected area of the elastic contact, the ch is the contact depth, the maxP is the maximum load upon loading, and the Among these samples, the multilayer inverse opals revealed shallower indentation depths as compared to those of single-layer inverse opals, suggesting the robust nature of inverse opals with gradient pores. From these load–displacement profiles, both hardness (quations .(1)A makes it feasible to form multilayer inverse opals with many possible new applications. We adopted a sequential electrophoresis technique to prepare multilayer colloidal crystals consisted of PS microspheres in size of 300, 600, and 1000 nm, respectively. The electrophoresis parameters were optimized so the larger PS microspheres were stacked atop of smaller ones in an orderly manner. The inverse opals with gradient pores were prepared by galvanostatic plating of Ni within the colloidal template, followed by the selective removal of PS microspheres. The SEM images exhibited ideal close-packed structures with well-defined interfaces among different layers. Results from PMI revealed a size of bottlenecks consistent with those of interconnected pore channels. Mechanical properties determined by nanoindentation tests indicated significant enhancement in hardness and moderate improvement in reduced elastic modulus from multilayer inverse opals as compared to those of single-layer ones. The increased mechanical stability favors further development of these nanostructured materials in practical applications, such as their direct utilization or the leverage in support materials.