extfieldsofstudy: Biology Medicine

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The Ontogeny of Cytochrome P450 Enzyme Activity and Protein Abundance in Conventional Pigs in Support of Preclinical Pediatric Drug Research

Since the implementation of several legislations to improve pediatric drug research, more pediatric clinical trials are being performed. In order to optimize these pediatric trials, adequate preclinical data are necessary, which are usually obtained by juvenile animal models. The growing piglet has been increasingly suggested as a potential animal model due to a high degree of anatomical and physiological similarities with humans. However, physiological data in pigs on the ontogeny of major organs involved in absorption, distribution, metabolism, and excretion of drugs are largely lacking. The aim of this study was to unravel the ontogeny of porcine hepatic drug metabolizing cytochrome P450 enzyme (CYP450) activities as well as protein abundances. Liver microsomes from 16 conventional pigs (8 males and 8 females) per age group: 2 days, 4 weeks, 8 weeks, and 6–7 months were prepared. Activity measurements were performed with substrates of major human CYP450 enzymes: midazolam (CYP3A), tolbutamide (CYP2C), and chlorzoxazone (CYP2E). Next, the hepatic scaling factor, microsomal protein per gram liver (MPPGL), was determined to correct for enzyme losses during the fractionation process. Finally, protein abundance was determined using proteomics and correlated with enzyme activity. No significant sex differences within each age category were observed in enzyme activity or MPPGL. The biotransformation rate of all three substrates increased with age, comparable with human maturation of CYP450 enzymes. The MPPGL decreased from birth till 8 weeks of age followed by an increase till 6–7 months of age. Significant sex differences in protein abundance were observed for CYP1A2, CYP2A19, CYP3A22, CYP4V2, CYP2C36, CYP2E_1, and CYP2E_2. Midazolam and tolbutamide are considered good substrates to evaluate porcine CYP3A/2C enzymes, respectively. However, chlorzoxazone is not advised to evaluate porcine CYP2E enzyme activity. The increase in biotransformation rate with age can be attributed to an increase in absolute amount of CYP450 proteins. Finally, developmental changes were observed regarding the involvement of specific CYP450 enzymes in the biotransformation of the different substrates.

INTRODUCTION
It has been about 20 years since the first pediatric legislations were initiated by the EMA and the FDA to stimulate pediatric drug research. Over the years this has led to an increased number of pediatric clinical trials, new medicines and pediatric indications both in the European Union and the United States (Califf, 2016;EMA, 2016). In order to support the safety of these pediatric trials, it is important to improve and optimize pediatric preclinical studies. Pediatric juvenile animal studies can provide essential information regarding the PK, PD and safety of a drug. According to the ICH guideline M3(R2), these studies should only be considered when previous animal data and human safety data are insufficient to support pediatric studies. If a juvenile study is warranted, only one relevant species, preferably rodent, is generally considered adequate. This is in contrast with preclinical studies in adults where both rodent and non-rodent species are mandatory (Food and Drug Administration, HHS, 2010). When selecting the appropriate animal species, it is important to take the maturation and growth of the different organ systems into account. From a historical point of view the rat is still the species of first choice when performing pediatric preclinical studies (Andrew et al., 2013). However, more recent reports suggest the pig (Sus scrofa domestica) as a valuable alternative animal model to the rat (Bode et al., 2010;Swindle et al., 2012;Helke and Swindle, 2013). Gasthuys et al. (2016) reported a high level of anatomical and physiological similarities between conventional pigs and humans during the developmental stages of life, rendering the piglet a suitable juvenile animal model. Nevertheless still a lot of knowledge gaps remain, such as the maturation of ADME processes both in growing children and piglets.
CYP450 enzymes are the most important phase I drug metabolizing enzymes, located on the smooth endoplasmic reticulum and highly expressed in liver compared to other tissues in pigs (Antonovic and Martinez, 2011;Nielsen et al., 2017).
They metabolize between 70 and 80% of the human drugs used, most frequently by hydroxylation (Matalová et al., 2016). The ontogeny of the human CYP450 enzymes is rather well known. For example CYP2C9 enzyme activity increases rapidly after birth reaching adult values around 3 months of age, whereas CYP1A2 reaches adult values around 10 years of age (Alcorn and McNamara, 2002;Johnson et al., 2006;Matalová et al., 2016). In contrast to men, the ontogeny of CYP450 enzymes in pigs remains largely unknown. Hu (2015) reported in Camborough-29 pigs an increase in average total CYP450 contents in liver microsomes and in biotransformation rate with age for six different substrates (phenacetin, coumarin, tolbutamide, bufuralol, chlorzoxazone, and midazolam) (Hu, 2015). Postnatal maturation of CYP450 enzyme activity in liver microsomes of the Göttingen minipig using four different substrates (phenacetin, tolbutamide, midazolam, and dextromethorphan) was reported by Van Peer et al. (2017). In general, CYP3A4, CYP2C9, and CYP2E1 are estimated to contribute to the metabolism of over 50% of human drugs currently on the market (Alcorn and McNamara, 2002). There is evidence that the three probe substrates, midazolam, tolbutamide, and chlorzoxazone, which are often used in the pharmaceutical industry for human in vitro work, are biotransformed to the same metabolites in pigs as in humans (Yuan et al., 2002;Helke and Swindle, 2013). The 1-hydroxylation of midazolam, 4-hydroxylation of tolbutamide, and 6-hydroxylation of chlorzoxazone have been used before as markers for, respectively, CYP3A, CYP2C, and CYP2E in pigs (Hu, 2015;Van Peer et al., 2017). Although high percentages of sequence identity of nucleotides and amino acids between pigs and humans are present, this is not always reflected in the substrate affinities (Puccinelli et al., 2011). For example, tolbutamide which is a selective substrate for human CYP2C9 shows some cross-reactivity in pigs, indicating that either the substrate is not specific or other enzymes are involved in the biotransformation (Skaanild, 2006).
When using microsomes to examine the biotransformation of drugs, the final goal is determining the intrinsic clearance and extrapolation to in vivo (IVIVE). This can be done by using hepatic scaling factors such as the MPPGL. In men, the MPPGL increases from birth (26 mg/g) to a maximum of 40 mg/g at approximately 28 years of age, followed by a gradual decrease with aging (Barter et al., 2007(Barter et al., , 2008. In pigs, this has only been done in two Suffolk White adult pigs obtaining values of 32.6 and 36.2 mg/g liver (Achour et al., 2011). Finally, the intrinsic clearance can be calculated with liver weight as a second scaling factor (Barter et al., 2007).
Another important factor in IVIVE is the CYP450 protein abundance. Barter et al. (2013) integrated specific abundances and phenotypes associated with several CYP450s in Caucasian and Chinese population models. This led to simulation of observed population differences in the clearance of several reference drugs. Regarding the pediatric population, and especially children younger than 2 years of age, Johnson et al. (2006) showed that physiologically based models resulted in better predictions than simple allometric scaling. The CYP450 protein abundances can thus support a better quantitative understanding of species differences and result in better scaling of PK and safety data to the clinical setting.
The aim of the current study was to gain more insight in the ontogeny of porcine CYP450 enzymes. First, the CYP450 enzyme activity was determined in hepatic microsomes using three probe substrates (midazolam, tolbutamide, and chlorzoxazone) in four age categories of growing piglets. Next to activity measurements, the MPPGL was determined in order to calculate the intrinsic clearance. Finally, the protein abundance of hepatic CYP450 in the microsomes was measured and compared with the enzyme activity.

Animals and Sample Collection
The current study was approved by the ethical committee of the Faculties of Veterinary Medicine and Bioscience Engineering of Ghent University (EC2016/105). Care and use of the animals was in full compliance with the national (Belgian Royal Decree of 29 May 2013) and European legislation on animal welfare and ethics (Anonymous, 2010/63/EU). Sixteen pigs (8 barrows and 8 sows, Hybrid sow × Piétrain boar) aged 2 days, 4 weeks, and 8 weeks were purchased from Gesuporc BVBA (Vorselaar, Belgium). Eight intact boars aged 6 months and eight sows aged 7 months (Large white × Land race, Seghers hybrid) were purchased from RA-SE Genetics and Convis (Ettelbruck, Luxembourg). The same strain of Seghers hybrids were used as the mothers of the younger age categories, thus reducing interindividual variability. The 2-day, 4-week, and 8-week males were castrated. However, the mean age for boars to reach puberty was reported to be at 21 weeks of age, while at 26 weeks of age only 8-31% of gilts were in puberty according to Van den Broeke et al. (2015). Therefore, no effect of castration (and thus sex hormones) is expected on the results. The four chosen age categories represent different groups of growing children. Two-day-, four-week-, eight-week-, and six-to-seven-month-old pigs correspond with neonates, infants, children, and adolescents, respectively (Shayne, 2015). The sows were 1 month older than the boars as females reach puberty later compared to males (Van den Broeke et al., 2015). All pigs were weighed (BW, kg) and euthanized at arrival at the test facility by intramuscular injection of a mixture of xylazine and tiletamine-zolazepam, followed by intracardial injection of a sodium pentobarbital overdose. The liver was excised, patted dry with compresses and weighed (LW, g). A piece of the right liver lobe (1.22-8.18 g) was snap-frozen in liquid nitrogen and stored at −80 • C until microsome preparation. CYP450 enzymes were stable in frozen liver samples for up to 4 months of storage time (Schelstraete et al., 2018).

Preparation of Microsomes
Microsomes were prepared as described by Goossens et al. (2013) using the differential ultracentrifugation method (Wilson et al., 2003). In short, liver samples were thawed on ice while soaking in homogenization buffer (0.25 M aqueous phosphate buffer containing 1.15% KCl, pH 7.25). The liver tissue was weighed, minced with a scissor and homogenized in 16 mL of homogenization buffer using a Potter-Elvehjem system (VWR). An aliquot of the homogenate was frozen (−80 • C) in order to determine the MPPGL (see section "Microsomal Protein per Gram Liver"). After centrifugation (Beckman L8-70M Ultracentrifuge, Beckman Coulter Limited, High Wycombe, United Kingdom) at 10,000 g for 25 min at 4 • C, the supernatant was centrifuged at 100,000 g for 80 min at 4 • C. The microsomal pellet was washed by resuspension in 16 mL of homogenization buffer, followed by centrifuging according to the latter conditions. The final microsomal pellet was resuspended in 1.5 mL/g liver tissue resuspension buffer (0.25 M aqueous phosphate buffer containing 1.15% KCl and 30% glycerol, pH 7.25). The microsomal suspension was snap-frozen in liquid nitrogen and stored at −80 • C until further analysis.

CYP450 Activity Measurements
Activity measurements were performed as described by Goossens et al. (2013). Total protein concentration in the microsomes was determined using the Bradford assay (Bradford, 1976). Three different probe substrates, namely midazolam, chlorzoxazone, and tolbutamide, were used to determine the CYP3A, CYP2E, and CYP2C enzyme activity, respectively. The formation rate of 1-hydroxy-midazolam, 6-hydroxy-chlorzoxazone, and 4-hydroxy-tolbutamide was analyzed, respectively. In order to optimize the ideal incubation conditions, the linearity, K M and maximum biotransformation rate (V max ) were determined on a pool of four 6 months old pigs (results not shown). An apparent K M of 13.7, 126.2, and 613.2 µM for midazolam, chlorzoxazone, and tolbutamide, respectively, was determined. The apparent V max was estimated at 281.3, 1210.1, and 153.3 µmol/min/mg microsomal protein, respectively. The incubation conditions were as follows. The preheated (37 • C) incubation mixture consisted of 50 µL of 1.15% KCl in water, 50 µL of 0.05 M aqueous phosphate buffer (pH 7.4), and 50 µL of substrate in water mixed with 50 µL NADPH (final concentration of 1 mM). After 3 min of pre-incubation, the enzymatic reaction was initiated by addition of 50 µL of microsome suspension. The microsomes were diluted in a 1.15% KCl solution, resulting in a final concentration of 0.25 mg/mL in the incubation mixture for all substrates. Midazolam (10 µM) was incubated for 10 min, while chlorzoxazone (100 µM) and tolbutamide (100 µM) were incubated for 20 min. After the specified incubation time, the enzymatic reactions were terminated by adding 25 µL of ice cold water/ACN/formic acid mixture [42/55/3, (v/v/v)], containing the internal standard chlorpropamide (final concentration of 0.072 µM). Next, the incubation mixtures were vortexed and transferred on ice. Subsequently, the samples were centrifuged at 20,000 g for 10 min (4 • C) after which the supernatant was collected and frozen at −20 • C until analysis. Each microsomal sample was incubated separately with the three substrates in triplicate. The metabolites formed during these incubations were quantified using a validated UHPLC-MS/MS method (De Bock et al., 2012).
The microsomal activity expressed as pmol/min/mg microsomal protein was corrected to nmol/min/g liver by means of the MPPGL (equation 1; section "Microsomal Protein per Gram Liver").

Microsomal Protein Per Gram Liver
The MPPGL of each pig (2-day-old ♂ = 2, ♀ = 8; 4 weeks, 8 weeks, and 6-7 months each time ♂ = 8, ♀ = 8) was calculated after determination of the microsomal recovery factor. This recovery factor was estimated by analyzing the NCR activity in homogenate and microsomes, according to Guengerich et al. (2009). Homogenate and microsomes were both diluted to 60 mg/mL (100 mg/mL for the 2-day-old piglets). 0.5 mM equine heart cytochrome c in 10 mM aqueous potassium phosphate buffer (pH 7.7) was prepared and frozen (−20 • C) until use. Nine-hundred microliters of 0.3 M potassium phosphate buffer (pH 7.7) containing 1 mM KCN, 80 µL of cytochrome c (0.5 mM) and 10 µL of the diluted homogenate or microsomes were mixed. After addition and mixing of 10 µL of reduced NADPH solution (10 mM in water), the absorbance was monitored at 550 nm for 4 min. The rate of cytochrome c reduction or NCR was calculated according to equation 2: A550/min/0.021 = nmol of cytochrome c reduced per minutein the cuvette (Eq. 2) The microsomal recovery factor was then calculated by the ratio of the NCR in the microsomes and the homogenate. Finally the MPPGL was calculated by the following equation (equation 3): MPPGL (mg/g) = yield of microsomal protein (mg/g)/ microsomal recovery factor (Eq. 3)

CYP450 Protein Abundancy Measurement: HD-DDA MS Experimental Set-up
Microsomal proteins (20 µg) of each individual pig were reduced in 0.5 M TEABC and 1 mM DTT for 1 h at 60 • C, followed by alkylation using 10 mM MMTS for 10 min at room temperature. Digestion of proteins into peptides was performed using trypsin (33:1 protein/enzyme ratio) overnight at 37 • C with CaCl 2 and ACN to a final concentration of 1 mM and 5%, respectively, as described earlier (Glibert et al., 2014). After evaporation, the samples were resuspended in 0.1% formic acid. Four hundred nanograms sample was spiked with 50 fmol beta-galactosidase and 50 fmol Hi3 E. coli standards before injection.
The peptides were separated using a nanoscale UPLC system (nanoACQUITY UPLC R , Waters, Milford, MA, United States) coupled to a Synapt G2-Si mass spectrometer (Waters). Peptides were first trapped in 0.1% formic acid on a 180 µm × 20 mm C18 Trap column. Separation was performed on a HSS C18 1.8 µm, 75 µm × 250 mm analytical column at a flow rate of 300 nL/min and a temperature of 45 • C. Mobile phase A and B were composed of, respectively, 0.1% formic acid with 4% DMSO in UPLC-water and 80% ACN containing 0.1% formic acid. Peptides were separated with a linear gradient for 60 min at 1-40% solvent B and for 1 min 40-85% solvent B. The Q-TOF Synapt G2-Si instrument was operated in positive mode for High Definition-DDA, using a nano-ESI source, acquiring full scan MS and MS/MS spectra (m/z at 50-5,000) in resolution mode. Survey MS scans were acquired using a fixed scan time of 200 ms. Tandem mass spectra of up to eight precursor ions with charge state 2+ to 5+ were generated using CID in the trapping region with intensity threshold set at 3,000 cps, using a collision energy ramp from 6/9 V (low mass, start/end) up to 147/183 V (high mass, start/end). MS/MS scan time was set to 100 ms with an accumulated ion count 'TIC stop parameter' of 100,000 cps allowing a maximum accumulation time of 250 ms. Dynamic exclusion of fragmented precursor ions was set to 12 s. Ion mobility wave velocity was ramped from 2,500 to 400 m/s for wideband enhancement to obtain a near-100% duty cycle on singly charged fragment ions. LockSpray of glufibrinopeptide-B (m/z at 785.8427) was acquired at a scan frequency of 60 s.
Data analysis of the raw files obtained from the Synapt G2-Si was performed in Progenesis TM QI (Nonlinear Dynamics) version 2.3. Peptides with charge +1 were discarded. For relative quantification, data was normalized to all proteins. For absolute quantification, data was normalized to Hi3 E. coli peptides. Peptide identification was performed with Mascot 2.5, the following search criteria were set: trypsin as digestion enzyme, up to two missed cleavages allowed, fixed modification of methylthio cysteine and variable modifications of methionine oxidation and deamidation at asparagine and glutamine. Peptide mass tolerance was set to 15 ppm and fragment mass tolerance to 0.2 Da. Protein identifications were obtained by searching a compiled database of reviewed Sus scrofa entries (SwissProt), supplemented with unreviewed CYP proteins and fragments of interest, the cRAP database (laboratory proteins and dust/contact proteins 1 ) and sequences of spiked standard proteins. For relative quantification, the top three peptides were used and only proteins with at least one unique peptide were further considered. For absolute quantification, proteins were quantified using the top three unique peptides against Hi3 E. coli peptides, and only proteins with at least one unique peptide were further considered. Protein data was exported from Progenesis TM for further statistical analysis.

Statistical Analysis
Sex and age differences in enzyme activity, MPPGL, and protein abundances were analyzed by a two-way nested ANOVA on the log-transformed data. Homogeneity of variances was evaluated using Levene's test. Tukey's honest significant difference test was performed post hoc. P-values equal to or below 0.05 were considered significant. A non-parametric Spearman rank correlation test was used to identify relationships between CYP450 protein abundancy and activity for each age category separately, and all ages together. CYP450 proteins with a correlation coefficient equal to or bigger than 0.7 were considered to be involved in the biotransformation of the substrate if more than seven correlations were statistically significant (p < 0.05). All analyses were performed in Rstudio (version 1.0.153, RStudio Inc, Boston, MA, United States). Finally, stepwise linear regression analysis was performed on the logtransformed data to evaluate allometric relationships for the intrinsic clearance (biotransformation rate for the full liver) and protein abundance of the involved CYP450 proteins as determined in the correlation analysis. Liver weight, body weight, and sex were the covariates of interest (SPSS 23, IBM, United States).

CYP450 Enzyme Activity
The microsomal enzyme activity (pmol/min/mg microsomal protein) increased with age for the three substrates (Supplementary Figure S1). No significant sex differences were found within the different age groups when the activity was expressed as per mg microsomal protein. This activity was corrected for enzyme losses during the fractionation process according to equation 1. The main effect of gender was significant (P < 0.05) for chlorzoxazone when the activity was converted to per g liver. Overall, the biotransformation rate for the three substrates increased with age, but differently according to the enzymes involved (Figure 1).

Microsomal Protein Per Gram Liver
The protein content estimated by the Bradford assay was corrected for protein loss using the microsomal recovery factor. The MPPGL calculated according to equation 3 is depicted in Figure 2. The main effect of gender within the age groups was significant (P < 0.05) and at 8 weeks of age sex differences were found to be significant. The youngest age category had a significant higher MPPGL compared to the other age categories. Significant differences were also observed between the 4-weekold pigs and the 8-week-old pigs and the 8-week-old pigs and 6-to 7-month-old pigs. No significant differences were observed between the 4-week-old pigs and the 6-to 7-month-old pigs.

CYP450 Protein Abundancy
In total 255 proteins were retrieved in the liver microsomes. Twenty CYP450 proteins were identified from which 12 had 3 or more unique peptides (Supplementary Table S1). All unique peptides were verified in Mascot. Two different CYP2E1 proteins were found which have a different protein existence. This is shown as a number after an underscore, namely CYP2E1_1 and CYP2E1_2. Number 1 means there is experimental evidence at protein level for the existence of the protein. The criteria include partial or complete Edman sequencing, clear identification by MS, X-ray, or NMR structure, good quality protein-protein interaction or detection of the protein by antibodies. The number 2 means there is evidence of protein existence at transcript level which means that the existence has not been strictly proven but that expression data (such as existence of cDNA(s), RT-PCR or Northern blots) indicate the existence of a transcript. Verification of these two proteins identified small differences in sequence, hence, these enzymes were considered as two different proteins. A relative quantification was performed to evaluate sex and age differences for every CYP450 enzyme found (Supplementary Figure S2). Significant sex differences in abundancy were only found in the 6-to 7-month-old pigs for CYP1A2, CYP2A19, CYP3A22, CYP2E1_1, CYP2E1_2, CYP4V2, and CYP2C36. The absolute quantification against Hi3 E. coli peptides was used to visualize the maturation of CYP450 enzymes in microsomes (Figure 3). The CYP450 proteins in the 6-to 7-month-old pigs were used as reference and are represented as a full pie of 100%. This abundancy was used to calculate the percentage CYP450 protein of the younger age categories. The individual percentages per age category can be found in Supplementary Table S2. A graphical overview of the absolute amount of CYP450 proteins (fmol) found in microsomes for every age category and per sex can be found in Supplementary Figure S3. In total 133.2 (±7.91), 435.2 (±30.83), 488.5 (±31.89), 683.3 (±47.67), and 1,186.8 (±108.11) pmol CYP450 proteins per mg microsomal protein were determined for 2-day-, 4-week-, 8-week-, 6-and 7-monthold-pigs, respectively. FIGURE 1 | The biotransformation rate of midazolam to 1-hydroxy-midazolam (A), tolbutamide to 4-hydroxy-tolbutamide (B) and chlorzoxazone to 6-hydroxy-chlorzoxazone (C), expressed as nmol per min per gram liver of conventional pigs (2-day-old ♂ = 2, ♀ = 8; 4 weeks, 8 weeks, and 6-7 months, each time ♂ = 8, ♀ = 8). No significant sex differences within the age groups were observed. Different letters indicate significant differences (P < 0.05) between the different age groups. The boxplots give the median, 25th and 75th percentiles. The upper and lower whisker extends from the hinge to the largest/smallest value, respectively, no further than 1.5 times the interquartile range. Data beyond the end of the whiskers are outliers and plotted individually.

Correlation CYP450 Protein Abundance Versus Enzyme Activity
The results of the Spearman rank test can be found in the correlation matrices presented in Supplementary Figure S4. To evaluate which enzyme might be involved in the biotransformation of the probe substrates over all age categories, a correlation coefficient of at least 0.7 was assumed when more than seven CYP450 proteins showed a statistically significant (P < 0.05) correlation. The results for each age category, and all ages together can be found in Table 1.

Regression Models
The log-transformed activity and abundancy data showed a linear relationship which is in support with the principle of allometric scaling. Both intrinsic clearance and protein abundance showed a better fit toward liver weight instead of body weight. Details on the best fits can be found in Table 2. Figure 4 gives an overview of the defined models with the corresponding observations. Additionally, the log ratio of the intrinsic clearance to the corresponding amount of biotransforming CYP450 proteins in the full liver was calculated. No correlation could be observed in function of the log liver weight.

DISCUSSION
Despite an increasing amount of data suggesting the piglet as a suitable animal model for pediatric preclinical studies, a lot of knowledge gaps still remain. Especially data on the ontogeny of the different organ systems involved in the ADME processes are lacking. This information is crucial to make reliable predictions of the behavior of a drug in growing children, based on juvenile animal data.

Microsomal Protein Per Gram Liver
One of the strategies to predict in vivo drug clearance in humans, is to extrapolate based on in vitro data. The initial step in this process is to convert in vitro microsomal enzyme activity expressed as units per milligram protein to per gram whole liver. This conversion is mostly done by a hepatic scaling factor such as the MPPGL. In humans, different adult values of 33, 40, and 52.9 mg/g liver were reported (Lipscomb et al., 2003;Wilson et al., 2003;Hakooz et al., 2006). The differences in adult MPPGL can be due to the liver source, disease state, and/or experimental methodologies. De Bock et al. (2014) measured an average value of 18.73 mg/g in four children with a mean age of 1.17 years suffering from biliary atresia. Barter et al. (2008) FIGURE 2 | Boxplot of the microsomal protein per gram liver (MPPGL) with increasing age of conventional pigs (2-day-old ♂ = 2, ♀ = 8; 4 weeks, 8 weeks, and 6-7 months, each time ♂ = 8, ♀ = 8). The boxplots give the median, 25th and 75th percentiles. The upper and lower whisker extends from the hinge to the largest/smallest value, respectively, no further than 1.5 times the interquartile range. Different letters indicate significant differences (P < 0.05) between the different age groups. The asterisk shows significant sex differences within the age group.
suggested increasing MPPGL values from birth (26 mg/g) to a maximum of 40 mg/g at around 28 years of age, followed by a gradual decrease when aging. However, it should be noted that the data from Barter et al. (2008) are based on a limited number of pediatric livers (11 fetal, age not provided; and 5 pediatric, mean age of 7.8 years). In the current study in pigs, the ontogeny of the MPPGL showed a different slope compared to the model of Barter et al. (2008). A decrease from 26.7 mg/g in neonatal piglets to 15.6, 11.7, and 14.4 mg/g at, respectively, 4 and 8 weeks and 2 6-7 months was demonstrated. Only one other study was found that determined the porcine MPPGL. Achour et al. (2011) reported a higher value of 32.6 and 36.2 mg/g liver in two Suffolk White adult pigs. These higher values compared to the values reported in the current study can be due to breed differences. Furthermore, the age was not exactly specified. To the authors knowledge, no study is available where the MPPGL is determined in growing piglets. In order to evaluate whether the porcine MPPGL can be extrapolated to humans, more pediatric data is mandatory in addition to the data provided by Barter et al. (2008).

CYP450 Enzyme Activity
The biotransformation rate of midazolam in pigs showed a steep increase during the first weeks of life followed by a gradual increase till 6-7 months of age. An increase in midazolam biotransformation rate with age was also observed by Hu (2015) in Camborough-29 pigs and by Van Peer et al. (2017) in Göttingen minipigs. In humans, midazolam is used as a selective probe for CYP3A4/5 enzyme activity. Several human models based on in vitro midazolam activity data have been proposed showing the maturation of CYP3A4 with age (Björkman, 2005;Edginton et al., 2006;Johnson et al., 2006). The human maturation curves are in line with the results found in the current study. Edginton et al. (2006) reported an enzyme activity greater than adult values around the first year or two of life. This higher activity is also observed in 4-week-old piglets compared to 8-week-old piglets (Figure 1).
The enzyme activity of tolbutamide in pigs, which is a specific human CYP2C9 substrate, increased gradually to a plateau with adult values reached at 8 weeks of age. Hu (2015) noticed a slightly different maturation curve in pigs, where no plateau was reached at 20 weeks of age. In humans, Johnson et al. (2006) reported a similar maturation curve as observed in the current study, where adult values are reached around 6 years of age. It should be mentioned that maturation curves in humans are based on a limited number of pediatric individuals and most liver samples are donated by diseased children, while all experiments in the current study were performed on healthy piglets.
The maturation curve for chlorzoxazone changed drastically after scaling with the MPPGL. This is in contrast to midazolam and tolbutamide, where no differences in maturation were observed whether the activity was expressed as pmol/min/mg protein or nmol/min/g liver. When expressed as pmol/min/mg microsomal protein, the chlorzoxazone activity gradually increased during the first 8 weeks of life, reaching a plateau at 6-7 months of age. When expressed as nmol/min/g liver, the activity remained constant during the first 8 weeks of life and increased when reaching puberty. This demonstrates the importance of a hepatic scaling factor taking the enzyme losses during microsome preparation into account, such as the MPPGL used here. Moreover, Zhang et al. (2015) found the CYP450 activity based on liver tissue to be superior to the CYP450 activity based on microsomal proteins in human. The maturation curve for human CYP2E1, as predicted by Johnson et al. (2006), is different than the maturation curve for the porcine chlorzoxazone biotransformation. This is probably due to the fact that chlorzoxazone is metabolized by more or other porcine enzymes than CYP2E1 (Wiercinska and Squires, 2010).

CYP450 Protein Abundancy
In order to identify which CYP450 enzyme is involved in the biotransformation of a certain substrate, a correlation analysis between the enzyme abundance in the microsomes and the activity was performed. It is assumed that the more enzyme is present, the higher the transformation rate will be. In literature, protein abundances are mostly determined via mRNA expression (PCR) or immunoquantification (Anzenbacher et al., 1998;Skaanild and Friis, 2008;Rasmussen et al., 2011). Disadvantages of these techniques are that due to posttranslational modifications, the amount of protein is not strictly regulated by the expression of mRNA. Also, the human or rat antibodies used for immunoquantification are prone to cross-selectivity. Furthermore, a selection of CYP450 enzymes of interest has to be made prior to the experiments. The latter is in contrast with the proteomics approach used in the current study, where the enzyme activity can be correlated with all CYP450 proteins found in the microsomes. Overall for midazolam, it was shown that CYP3A46, CYP3A22, and CYP3A are involved in the biotransformation to 1-hydroxymidazolam when all pigs of all age categories were analyzed FIGURE 3 | Ontogeny of CYP450 enzymes in growing conventional piglets (2-day-old, 4 weeks, 8 weeks and 6-7 months, each time ♂ = 8, ♀ = 8). Six to seven months old pigs are used as reference and CYP450 abundancy is represented as a full pie of 100%. Since this is the only age category where significant sex differences were found, separate charts for male (D) and female (E) are shown. For the 2-day-(A), 4-week-(B) and 8-week-old (C) piglets, the average of both sexes is presented.
together. These CYP450 show a high percentage of identity with their equivalent human CYP450 enzymes, CYP3A4/5/7/43 (Supplementary Table S1). Midazolam could thus be considered as a selective porcine substrate to evaluate the CYP3A enzyme activity. However, when looking at the individual age categories it seems that only at 6-7 months of age CYP3A22 showed a high correlation with midazolam biotransformation. In the younger age categories other CYP450 proteins seem to be involved in the biotransformation. This might be an indication that during development other CYP450 enzymes take over when the CYP450 of interest is not yet abundant enough. For example, van Waterschoot et al. (2008) observed in CYP3A knockout mice midazolam biotransformation by upregulated CYP2C enzymes. Regarding tolbutamide, CYP2C34, CYP2C35, CYP2C36, and CYP2C49 are involved in its biotransformation when evaluating all age categories together. These porcine CYP450 enzymes have 73.1-80.6% of identity with human CYP2C8/9/18/19, suggesting tolbutamide to be a good substrate to evaluate porcine CYP2C activity. The CYP2C family is involved in the tolbutamide biotransformation from 2-day-old piglets onwards, but at 6-7 months of age it appeared that CYP2B22 and CYP51A1 also contributed to the biotransformation. It was already reported that chlorzoxazone might be a poor substrate to evaluate CYP2E activity in pigs (Skaanild, 2006;Wiercinska and Squires,  Each age category consists of 8 males and 8 females. / No statistically significant correlation coefficient was found. PE, protein existence, these are two different CYP2E1 proteins. TABLE 2 | Properties of the different regression models regarding intrinsic clearance for midazolam, tolbutamide, and chlorzoxazone, the amount of CYP450 proteins in the liver for the biotransformation of the corresponding probe substrate and liver and body weight. When the effect of gender was significant (P < 0.05) this was included in the equation. LW, liver weight (g); BW, body weight (kg); Intr Cl, intrinsic clearance; MDZ, midazolam; TB, tolbutamide; CZ, chlorzoxazone; CYP450, cytochrome P450; ns, not significant; SE, standard error.

2010
). This could be confirmed in our study where CYP3A46, CYP3A, and CYP2C33v4 showed the highest correlation with chlorzoxazone biotransformation when all pigs were considered. However, the selectivity of involved CYP450 varied with age.
In 2-day-old piglets, CYP2C34 was the CYP450 of interest, while at 8 weeks of age CYP2A19 seemed to be involved in the biotransformation of chlorzoxazone. Finally, at 6-7 months, CYP1A2, CYP2A19, CYP3A, CYP3A22, CYP2E1_1, CYP2E1_2, and CYP4V2 showed a significant correlation. Wiercinska and Squires (2010) found chlorzoxazone to be metabolized by CYP1A1, CYP2E1, CYP2A19, and CYP2C33v4 which is in line with the work presented here. However, it seems that age has an influence on the specific CYP450 involved. The current study also showed a significant correlation between CYP3A22, CYP3A46, and CYP3A on the one hand, and chlorzoxazone on the other hand. This is in contrast with Wiercinska and Squires (2010) were no evidence of chlorzoxazone biotransformation by CYP3A was found. The CYP3A involvement might be due to the high correlation between CYP450 proteins mutually. A high correlation coefficient doesn't necessarily means that the CYP450 of interest is also involved in the corresponding biotransformation. Ideally, all selected CYP450 proteins should be incubated individually with the substrates to evaluate their contribution. Nevertheless, the use of chlorzoxazone as a porcine CYP2E substrate is not advised. It should be noted that at 4 weeks of age no significant correlations could be found for midazolam or chlorzoxazone. CYP3A22 and CYP3A46 had the highest coefficient of 0.47 and 0.35, respectively, with midazolam. Chlorzoxazone showed the best correlation with CYP2A19, CYP4A, and CYP4A24 with a coefficient of 0.41, 0.46, and 0.46, respectively. Further research is required to evaluate the age-related involvement of different CYP450 proteins in the biotransformation of the corresponding substrates. Recently, in our research group, Schelstraete et al. (2018) included two more probe substrates to gain more insight in the selectivity of porcine CYP450 enzymes (submitted). There is ample evidence available that the increase in biotransformation rate can be attributed to developmental expression of CYP450 mRNA and proteins in humans and pigs (Alcorn and McNamara, 2002;Van Peer et al., 2015) The current study supports this hypothesis as an increase in biotransformation rate with age can be related to an increase in absolute amount of CYP450 proteins in the microsomes. At 2 days of age only 14% of CYP450 proteins are present compared to the 6-to 7-month-old pigs, which is reflected in a very low biotransformation rate of the three substrates. Fourand eight-week-old piglets have around 50% of CYP450 proteins compared to the 6-to 7-month-old pigs. At 6-7 months of age, the amount of CYP450 proteins is considered to be at maximum adult values with 100% CYP450 proteins present in the liver. Achour et al. (2011) was able to identify 10 CYP450 proteins in microsomes of two Suffolk white adult pigs. Both CYP2D6 and CYP2A19 were most abundant, which is in line with the results from the current study with an abundancy of 31 and 12% for males and 22 and 39% for females for CYP2D6 and CYP2A19, respectively. Achour et al. (2011) did not report any sex differences, as the study was only done in two pigs. Significant sex differences in activity and mRNA expression for CYP1A, CYP1A2, and CYP2A were observed by Rasmussen et al. (2011) in intact crossbreeds between Landrace × Yorkshire sire and Duroc boars of 164 days old. The CYP2E1 activity was higher in females compared to males, although not significant. Despite the higher activity and mRNA expression in female pigs, Rasmussen et al. (2011) did not found elevated levels of protein with western blotting in females. This is in contrast with the results presented here, where the 7 months old females have significantly higher CYP1A2, CYP2A19, CYP3A22, CYP4V2, CYP2E1_1, and CYP2E1_2 protein abundances. Higher female protein concentrations were also observed by Skaanild and Friis (1999) for CYP1A2 and CYP2E1 in both Göttingen minipigs and Danish Landrace × Yorkshire × Duroc cross-breed pigs (Skaanild and Friis, 1999). The 6-month-old males in the current study showed significant higher CYP2C36 protein abundance compared to females. The observed sex differences could be attributed to sex hormones. Van den Broeke et al. (2015) observed in 264 conventional pigs (Hybrid sow × Piétrain boar) that at 5 months of age, all boars reached puberty, but only 8-31% of gilts were in puberty. Therefore, in this study, gilts of 7 months old were included to represent sows in puberty. In order to evaluate whether these sex differences in CYP450 protein abundance have an influence on the biotransformation of drugs, selective substrates should be chosen to perform the same activity measurements as described above for midazolam, tolbutamide, and chlorzoxazone. In the current study, no significant sex differences in enzyme activity were found for these substrates. Although, this could have been expected for tolbutamide as CYP2C36 is involved in its biotransformation. This could possibly mean that other CYP450 enzymes contributed more to the biotransformation of tolbutamide than CYP2C36.
In humans, several studies have been performed to determine the protein abundance of the CYP450 enzymes in adult microsomes. This resulted in great variations of reported values. For example, Gröer et al. (2014) reported CYP3A4 abundance of 32.6 pmol/mg microsomal protein while Achour et al. (2017) measured 80.9 pmol/mg microsomal protein. A possible explanation could be the different methods used, rendering it difficult to compare the absolute porcine abundances with human. A better way to make a correlation between these two species is to compare the relative abundances. CYP2D6 (26%) and CYP2A19 (29%) are the most abundant CYP450 proteins in 6-to 7-month-old pigs, followed by CYP2E (13%) and CYP2C (18%) proteins. In humans, however, CYP2C9 (20%) is found to be the most prominent CYP450 protein in microsomes, followed by CYP3A4 (16%), CYP2E1 (13%), and CYP2A6 (12%) (Kawakami et al., 2011). Unfortunately, data regarding the ontogeny of the human CYP450 proteome is limited. One study reported increasing levels of CYP2B6 with age measured using immunoquantification (Pearce et al., 2016). Sadler et al. (2016) performed a more thorough comparison of CYP450 protein expression and activity profiles through human development using activity-based protein profiling. The porcine homologs of the human CYP450 enzymes marked by a postnatal increase, namely members of the CYP1A, 2A, 3A, 2C, 2D, and 2E subfamilies, also increased in protein amount in the current study. Sadler et al. (2016) however, did not found a direct correlation between CYP450 activity and protein expression. This is in contrast with the results presented here where high correlation coefficients were found between the biotransformation rate and certain CYP450 proteins. Further research is required to evaluate whether pediatric microsomes show a similar trend as seen in piglets, namely an increase in amount of CYP450 proteins which can be correlated with an increase in activity.
The biotransformation rate, as well as the amount of involved CYP450 proteins showed a log-linear relationship with the log of the liver weight (R 2 ≥ 0.83) (Figure 4). In addition, the log-ratio of the biotransformation rate to their corresponding CYP450 proteins showed no linear correlation in function of the log liver weight. This indicates that when the CYP450 protein is present, it will contribute to the biotransformation of the probe substrates regardless of the age or liver weight of the animal. The developmental biotransformation rates observed in the present study might thus be solely due to an increase in absolute amount of CYP450 proteins. Further research is, however, required to unravel the mechanisms regulating CYP450 enzyme ontogeny.

CONCLUSION
The ontogeny of porcine CYP450 enzymes was established on three levels, i.e., enzyme activity with and without hepatic scaling factor and protein abundance. The increase in biotransformation rate with age could be attributed to an increase in absolute amount of CYP450 proteins. Midazolam and tolbutamide were shown to be selective probe substrates to evaluate both human and porcine CYP3A and CYP2C enzyme activities, respectively. Chlorzoxazone is not advised to use as a selective porcine CYP2E substrate. This study provides more insight in the ontogeny of CYP450 mediated phase I metabolism in pigs, which can be used to support pediatric preclinical research. Further research is required to evaluate age-related changes in CYP450 biotransformation involvement and the regulating developmental mechanisms. Also, other relevant CYP450 enzymes and substrates should be evaluated.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
JM, EGa, LDB, JVB, DD, SC, MD, EGo, and LDC contributed conception and design of the study. JM organized the database and performed statistical analysis. SS performed linear regression analysis. JM, EGa, WS, EGo, and LDC aided in the acquisition of the work. JM wrote the first draft of the manuscript. EGo and LDC wrote a section of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.

FUNDING
This study was funded by the Agency for Innovation by Science and Technology in Flanders and the Agency for Innovation and Entrepreneurship in Flanders (IWT, SB141427).


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Recent advances in understanding lymphangiogenesis and metabolism

The blood and lymphatic vasculatures are vital to the maintenance of homeostasis. The interaction between two vascular networks throughout the body is precisely controlled to enable oxygen and nutrient delivery, removal of carbon dioxide and metabolic waste, drainage of interstitial fluid, transport of immune cells, and other key activities. Recent years have seen an explosion of information dealing with the development and function of the lymphatic system. The growth of lymphatic vessels, termed lymphangiogenesis, is a high-energy requirement process that involves sprouting, proliferation, migration, and remodeling of lymphatic endothelial cells and capillaries. Although there has been substantial progress in identifying growth factors and their downstream signaling pathways that control lymphangiogenesis, the role of metabolic processes during lymphangiogenesis and their links to growth factor signaling are poorly understood. In this review, we will discuss recent work that has provided new insights into lymphatic metabolism and its role in lymphangiogenesis.

Introduction
Adenosine 5'-triphosphate (ATP) is the main carrier of cellular energy and its generation is tightly coupled to metabolic demand, ensuring sufficient energy for cellular growth, repair, movement, and other biological processes 1 . The three principal sources used by mammalian cells to generate ATP molecules are fats, proteins, and carbohydrates 2 . These three nutrients are referred to as "mitochondrial nutrients" because they are essential commodities to yield ATP in mitochondria 3 . This process of generation of ATP in the mitochondria is referred to as oxidative phosphorylation, since it involves phosphorylation of ADP (to produce ATP) as a result of electron transfer from NADH or FADH 2 to O 2 by a series of oxidative reactions 4 . The end product is the generation of 32 ATP molecules from one molecule of glucose 5 . In addition to oxidative phosphorylation, ATP also can be generated anaerobically by glycolysis. During glycolysis, one molecule of glucose is converted into two molecules of lactate with the production of two molecules of ATP 6 . Traditionally, it has been assumed that normal cells rely predominantly on mitochondrial oxidative phosphorylation to yield most of the ATP needed for cellular work 7 . As will be described below, the situation in the endothelium, both blood and lymphatic, is quite different.
The lymphatic vasculature serves a number of key functions, including regulation of interstitial fluid homeostasis and immune cell transport 8 . The majority of lymphatic endothelial cells (LECs) are derived from the anterior cardinal vein endothelial cells (ECs) around embryonic day 9.5 9,10 , although there are alternative sources for LECs in the skin 11 , heart 12 , and intestine 13 . During lymphangiogenesis, the primary lymphatic plexus is reorganized into lymphatic capillaries, lymph nodes, and collecting lymphatic vessels 14,15 . Lymphatic capillaries serve as entry points for the interstitial fluid that then is transported, via collecting lymphatic vessels, back to the blood circulation via the thoracic duct that drains into the left subclavian vein. Similarly, immune cells enter the lymphatics via its open-ended capillaries and then are transported to regional lymph nodes 16 . Not surprisingly, all processes involved in lymphatic vasculature specification, formation, and maintenance, as well as performance of normal everyday activities, require energy. Recent advances have led to a better understanding of lymphatic metabolism and their contribution to lymphangiogenesis, which is the subject of this review.

Metabolic environment of lymphatic endothelial cells
The lymphatic endothelium exists in a unique metabolic environment. The lymphatic fluid is rich in nutrients, even though it is the product of drainage of fluid waste from peripheral tissues. Typically, LECs are exposed to high concentrations of glucose (4-6 mM) 17,18 , total protein (8-32 g/L) 17 , and triglycerides (8-40 mg/dL) 17,18 . At the same time, oxygen concentration in the lymphatic fluid in the thoracic duct and capillary lymphatics is relatively low with partial pressure of oxygen (pO 2 ) 15-42 mmHg in the lymph fluid compared with 80-100 mmHg in the arterial blood [19][20][21][22] . This environment may, to an extent, account for the metabolic peculiarities of the lymphatic endothelium.

Glucose metabolism in lymphatic endothelial cells
Glucose is the principal energy source in the endothelium 23 . Having entered the cell, it can undergo either oxidative phosphorylation, a catabolic process involving its conversion into carbon dioxide and water, or anaerobic glycolysis that leads to the production of lactate. When coupled with mitochondrial respiration, one molecule of glucose produces 32 ATP molecules upon complete oxidation 24 (Figure 1). Alternatively, anaerobic glycolysis, which takes place in the cytosol and does not involve the consumption of oxygen, leads to the production of two molecules of ATP during glucose-to-lactate conversion ( Figure 1). Initially, it was thought that most normal, healthy cells predominantly employ glucose oxidation rather than glycolysis and that the latter is reserved for anaerobic conditions 7,25-27 . However, many types of cancer cells as well as certain normal cell types, including astrocytes 27,28 , fibroblasts 29,30 , activated T cells 31 , and pro-inflammatory macrophages 31 , depend predominantly on glycolysis for their energy demands.
More recently, several studies documented that blood vasculature ECs also rely primarily on anaerobic glycolysis for their energy generation 32-34 . Studies of glycolytic flux using radiolabeled glucose show that 99% of glucose is converted to lactate, which is a final product of glycolysis, and only a very small fraction (~0.04%) of pyruvate enters the mitochondria for glucose oxidation in rat coronary ECs 32 . LECs have also been reported to take up massive amounts of glucose compared with non-endothelial healthy cells and to use glycolysis as the primary source of ATP (~85%) with mitochondrial glucose oxidation almost completely inactive 23 . One potential explanation for this preference is that anaerobic glycolysis allows the production of energy at sites of cell locomotion (that is, lamellipodia and filopodia formation), thereby avoiding the need for ATP transport from the mitochondria 35 . Another is that interstitial tissue fluid which is absorbed into lymphatic vessels is the end product of somatic oxidative metabolism with low oxygen concentration [19][20][21][22] , and glycolysis maximizes the efficiency for ATP synthesis under their distinct nutritional environment.

Fibroblast growth factor signaling and glucose metabolism
As its name indicates, fibroblast growth factors (FGFs) were first identified as regulators of fibroblast proliferation 36,37 . Over the past four decades since the first purification and identification of FGFs, it has become clear that FGF signaling is not limited to fibroblasts but occurs in most cell types. Furthermore, it is involved with numerous normal 38 and pathological conditions ranging from development 39-41 and tissue homeostasis to coronary artery disease 42 , scleroderma 43 , hypercholesterolemia 44 , pulmonary arterial hypertension 45,46 , and atherosclerosis 47,48 , among many others.
Recent studies demonstrated an important role played by the FGF signaling cascade in lymphatic development that, in many ways, is as critical and important as a better recognized contribution of vascular endothelial growth factor (VEGF) signaling 49-53 . FGF ligands initiate their signals through high-affinity Figure 1. Adenosine 5'-triphosphate generation in mammalian cells. Amino acids (glutamine), fatty acids (palmitic acid), and carbohydrates (glucose) are the main sources of adenosine 5'-triphosphate (ATP) in the mammalian cell. Glutamine enters the mammalian cell through a glutamine transporter such as ASCT2. Glutamine serves as a precursor for the synthesis of lipids, nucleotides, proteins, and ATP. Fatty acids (palmitic acid) are transported into cytosol by FATPs (fatty acid transporters) and collapsed into acetyl-CoA through FAO (fatty acid oxidation). CPT1 (carnitine palmitoyltransferase I) on the mitochondrial outer membrane and CPT2 (carnitine palmitoyltransferase II) on the inner membrane cooperate for FAO. During glucose oxidation, glucose is transported across the cell membrane by GLUTs (glucose transporters) and catabolized into pyruvate. Pyruvate then enters the mitochondria and is converted into acetyl-CoA, which initiates the tricarboxylic acid (TCA) cycle to generate ATP. During anaerobic glycolysis, glucose is catabolized into pyruvate by using the same mechanism of glucose oxidation, but pyruvate turns into lactate and is finally exported into the bloodstream by monocarboxylate transporter (MCTs). During this metabolic process, complete oxidation of one molecule of nutrient generates 106 ATP (palmitic acid), 32 ATP (glucose; aerobic), and 2 ATP (glucose; anaerobic) molecules. ATP, adenosine 5'-triphosphate; ETC, electron transport chain.
tyrosine kinase receptors (FGFR1-4) and a number of coreceptors, including syndecan-4 and Klotho 54 . There is a high level of redundancy between the 22 FGFs and four FGFRs, leading to frequent compensatory effects. One example of that is the paucity of vascular developmental abnormalities in Fgfr1 or Fgfr3 single-knockout 55 and Fgfr1/Fgfr2 double-knockout 56 mice. These phenotypes come to the fore, however, in mice with disrupted expression of both Fgfr1 and Fgfr3 genes (Fgfr1 fl/fl ; Fgfr3 −/− with Prox1[BAC]-CreER T2 ) 55 . These animals show impairment of LEC migration and lymphatic vessel branching, resulting in significant edema, appearance of blood-filled lymphatics, and reduced dermal lymphatic development 55 .
RNA-seq analysis of gene expression in LECs before and after FGF2 or small interfering RNA (siRNA)-mediated disruption of FGF signaling showed that FGFs regulate not only proliferation and migration but also metabolic processes in LECs, glucose metabolism in particular 55 . Among the genes involved in glucose metabolism, HK2 (hexokinase 2) was identified as the highest-ranking gene regulated by FGFs 55 .
Hexokinases (HKs) are rate-limiting enzymes catalyzing the first step of glucose breakdown, which is the ATP-dependent phosphorylation of glucose to glucose 6-phosphate (G6P) (Figure 2). Among their four isoforms (HK1-HK4), HK2 is a predominant isoform in insulin-sensitive cells such as cardiomyocytes, skeletal muscle, and adipocytes; it is also upregulated in many types of tumors, and its expression is associated with enhanced glycolysis and tumor growth 57 . HK2 deletion in either all or lymphatic-only ECs in mice using, respectively, Cdh5(PAC)-CreER T2 or Prox1(BAC)-CreER T2 showed reduced glycolysis and retarded blood or lymphatic vascular development (or both) in various organs, including skin, cornea, and retina 55 , supporting the importance of HK2.
One of the factors that regulate HK2 expression is hypoxia. Under hypoxic conditions, hypoxia-inducible factor-1 alpha (HIF1α) expression is increased and, in turn, enhances the expression of the HK2 gene through binding to the E-box sequence 5'-CACGTG-3' in the HK2 promoter in cooperation with MYC transcription factors 58,59 . In cell types dependent on oxidative phosphorylation, this mechanism can result in a shift to anaerobic glycolysis, thereby allowing the cells to survive in hypoxic conditions 58,59 . Interestingly, activation of FGF signaling in LECs also enhances their glycolytic flux through MYC transcription factor expression 55 (Figure 2). Reversely, suppression of FGF signaling input, including FGFR1 siRNA treatment or Fgfr1/Fgfr3 deletion in LECs, leads to reduced expression of MYC and HK2 and a suppression of glycolysis 55 (Figure 2). The importance of this "metabolic" aspect of FGF signaling is emphasized by the fact that an LEC-specific deletion of HK2 leads to a very similar phenotype with Fgfr1/Fgfr2 doubleknockout mice 55 . That the metabolic process could be manipulated through external growth factor-initiated signaling in LECs is intriguing because it suggests that the metabolic pathway could be a therapeutic target for lymphangiogenesis-related diseases.

Fatty acid oxidation in lymphatic endothelial cells
Fatty acids are an important nutrient source not only for energy generation but also for biomass synthesis necessary to support cell proliferation. Carbon and nitrogen derived from fatty acid oxidation (FAO) become the building blocks for nucleotide synthesis, which is necessary for DNA replication and transcription. Although in most cell types glucose and glutamine supply most of the carbon and nitrogen necessary to support cell growth and division 25 , recent studies demonstrated that in ECs fatty acid carbon, and not glucose, is primarily used to support RNA and DNA nucleotide synthesis 60 (Figure 3). Using radiolabeled palmitate, a 16-carbon saturated fatty acid, Schoors et al. showed that its carbon incorporates into aspartate (a nucleotide precursor) and uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates and DNA) in human Figure 2. Fibroblast growth factor signaling and lymphatic endothelial metabolism. After fibroblast growth factor (FGF) ligand binding, FGFR1 and FGFR3 initiate a signaling cascade and induce MYC expression in LECs. MYC binds to the regulatory region of the HK2 gene, a rate-limiting enzyme catalyzing the first step of glucose breakdown into glucose-6-phosphate. The FGFR1/3-MYC-HK2 axis is a crucial driver of glycolysis for LECs to generate adenosine 5'-triphosphate (ATP), which is needed for lymphangiogenesis. FGFR, fibroblast growth factor receptor; GLUT, glucose transporter; HK2, hexokinase 2; LEC, lymphatic endothelial cell. umbilical vein ECs 60 ( Figure 3). CPT1A (carnitine palmitoyltransferase 1A), an enzyme localized to the mitochondrial outer membrane, controls the translocation of fatty acids across the mitochondrial membrane and acts as the rate-limiting regulator of FAO. An endothelial-specific knockout of Cpt1a in mice (Cpt1a fl/fl ; Cdh5[PAC]-CreER T2 ) displayed reduced proliferation in stalk ECs, while leading tip cells are intact 60 , suggesting that FAO stimulates vessel sprouting through EC proliferation. Analogous to blood ECs (BECs), LECs also use fatty acid as the principal source of carbon for nucleotide biosynthesis, but the FAO flux in LECs is even higher than in BECs while glycolytic flux is lower, implying that LECs use a metabolic pathway distinct from that of BECs 61 .

Molecular signaling and fatty acid metabolism
A critical step in lymphangiogenesis is the specification of lymphatic endothelial fate. During embryonic development, around embryonic day 9.5, a small subset of anterior cardinal vein ECs begin expressing a lymphatic marker, Prox-1, which is driven by ERK-dependent activation of transcription factor Sox18 expression 15,62 . This leads to venous-to-endothelial cell fate transition and activation of expression of LEC-specific markers, including LYVE-1, PROX1, FGFR3, and VEGFR3 62,63 . PROX1-expressing LECs migrate into the surrounding tissue to form primitive lymph sacs and lymphatic plexus 15 . PROX1 regulates the lymphangiogenic receptor tyrosine kinase VEGFR3 expression, which is responsible for LEC migration and proliferation. The absence of PROX1 expression results in the complete loss of the lymphatic system and decreased VEGFR3 expression in developmental lymphangiogenesis 15 .
In addition to the PROX1-mediated molecular signaling pathway, a recent study showed that PROX1 is involved with FAO metabolism for lymphangiogenesis. In PROX1-deficient cultured LECs, CPT1A expression and FAO flux are also inhibited, and this led to inhibition of proliferation. Consistent with these findings, LEC-specific Cpt1 deletion in developmental mouse embryo (Cpt1 fl/fl with Prox1[BAC]-CreER T2 ) also showed defective edema caused by impaired LEC proliferation, migration, and sprouting 61 . Interestingly, FAO inhibition through CPT1KD decreased VEGFR3 expression, which suggests that FAO is essential not only for fatty acid metabolism but also for maintaining LEC identity through transcriptional gene regulation. Mechanistically, overexpression of PROX1 upregulates CPT1A transcription, which increases the generation of acetyl-CoA through fatty acid β-oxidation 61 (Figure 3). Acetyl-CoA In lymphatic endothelial cells (LECs), fatty acid is used primarily for nucleotide biosynthesis. During the tricarboxylic acid (TCA) cycle in mitochondria, acetyl-CoA provides the carbon skeletons for aspartate and uridine monophosphate. These intermediates are used in nucleotide synthesis in proliferating LECs. Acetyl-CoA catabolized from fatty acid is also used for histone acetylation of VEGFR3 promoter region with PROX1/P300 protein complex. (Left) The major source of ATP in LECs is glucose. (Right) Up to 85% of ATP is generated using glycolysis (anaerobic). ATP, adenosine 5'-triphosphate; CPT1A, carnitine palmitoyltransferase 1A; F1,6P, fructose 1,6-diphosphate; F6P, fructose-6-phosphate; FAO, fatty acid oxidation; FATP, fatty acid transporter; G3P, glucose 3-phosphate; G6P, glucose 6-phosphate; GLUT, glucose transporter; VEGFR3, vascular endothelial growth factor receptor 3.
is used for two different purposes on LECs. First, it provides a carbon source for nucleotide synthesis, which eventually leads to LEC proliferation 61 . Second, LEC uses acetyl-CoA for histone acetylation of VEGFR3 promoter region with PROX1 and p300 (histone acetyltransferase p300) protein complex 61 (Figure 3). Prox1 has also been shown to function as a metabolic transcriptional regulator in non-ECs. In liver cells, Prox1 interacts with ERRα/PGC-1α complex, which plays a central role in the maintenance of energy homeostasis and inhibits its activity 64 . In colon cancer cells, Prox1 controls metabolic adaptation of these cells, promotes resistance to nutrient deprivation, and fuels metastatic outgrowth of tumors 65 . With a chip-on-chip assay with Prox1, Prox1 was shown to target genes highly enriched for processes linked to bile acid biosynthesis and histidine metabolism 64 . This suggests that metabolic regulation might be one of the major activities of Prox1.
VEGFR3 is a critical and indispensable regulator of lymphatic development and function. Its almost exclusive expression in LECs makes them respond to vascular endothelial growth factor-C (VEGF-C) for LEC proliferation, migration, and sprouting 66 . VEGFR3 expression is known to vary with developmental stage and pathological conditions. It is expressed in BECs during early embryogenesis, but later, VEGFR-3 expression becomes restricted to the LECs. Vegfr3 knockout mice die because of angiogenic defect around embryonic day 10.5, before the establishment of the lymphatic system 67 . Heterozygous functional null mutations in the Vegfr3 gene which inactivate the tyrosine kinase do not cause any defect in embryonic angiogenesis, but they have been linked to lymphedema 68,69 . Dynamic changes in the endogenous expression level of VEGFR3 indicate that VEGFR3 transcription is precisely regulated. VEGFR expression can be altered by numerous transcription factors, including SP1, SP3, ETSs, p300, PROX1, and E2F 70-72 , and by epigenetic modification of the regulatory sequences, including histone acetylation and CpG methylation 72,73 . Among the genes mentioned, ETSs, Prox1, and p300 are involved in histone acetylation of the Vegfr3 gene, which likely plays a role in its transcriptional regulation 70-72 . This combination of events leads to fatty acid-derived acetyl-CoA, which plays a role in histone acetylation on the VEGFR3 promoter and regulation of VEGFR3 expression. Intriguingly, supplementation of acetate (a precursor of acetyl-CoA) is able to rescue lymphangiogenesis induced by CPT1A inhibition, implying the potential of metabolites for use in the field of regenerative medicine 61 . Although FAO plays crucial roles during the lymphangiogenesis process, it is not yet clear why FAO serves as the main carbon source for nucleotide synthesis in LECs even though the lymph fluid provides abundant nutrients, including glucose and amino acids 17,18 , which are employed for biomass synthesis in other tissues and whether FAO-derived acetyl-CoA also serves for acetylation modification of other lymphatic genes or proteins.

Perspective
In 1996, Joukov et al. purified and identified a cDNA that has VEGFR3 binding affinity and named it VEGF-C 74 . Over the past two decades since the identification of VEGF-C, several additional molecular pathways, including transcription factors, receptors, and mediators, have been shown to regulate lymphatic differentiation and lymphangiogenesis. In recent years, different lines of work have led to the conclusion not only that the molecular signals are important to control lymphangiogenesis but also that metabolism could be an alternative target. More detailed understanding of the metabolic pathway in LECs will open new avenues for developing new therapeutic targets for lymphangiogenesis-related diseases.

Competing interests
The author(s) declared that they have no competing interests.

Grant information
This work is supported by National Institutes of Health grants P01 Hl107205, HL084619 and HL135582.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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extfieldsofstudy: Chemistry Medicine

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Highly Bactericidal Macroporous Antimicrobial Polymeric Gel for Point-of-Use Water Disinfection

Access to clean and safe water supply remains inadequate in many developing countries. One of the key challenges is to remove pathogenic bacteria from the water supply via effective water disinfection technologies to prevent the spread of diseases and to ensure the safety of consumers. Herein, a highly effective point-of-use (on-demand) water disinfection technology, in the form of a polymeric scaffold called macroporous antimicrobial polymeric gel (MAPG), is demonstrated. MAPG is easy to fabricate, completely organic and possess inherent antimicrobial property which makes it non-reliant on inorganic compounds such as silver where the long-term toxicity remains unknown. MAPG is highly bactericidal and can disinfect bacteria-contaminated water (ca. 108 CFU mL−1) at a capacity of about >50 times the mass of the organic material used, inactivating >99% of both Gram-negative and Gram-positive bacteria including Escherichia coli, Vibrio cholerae and Staphylococcus aureus within 20 minutes of treatment. When fabricated in a syringe, MAPG eliminates E. coli from contaminated water source by >8.0 log10 reduction in bacteria counts (i.e., no viable bacteria were detected after treatment), and the syringe can be reused multiple times without losing potency. The MAPG technology is not only restricted to water disinfection but may also be applicable in other bacteria inactivation applications.

In this study, we report the development of a novel polymeric material which possess excellent inherent antimicrobial activity, termed macroporous antimicrobial polymeric gel (MAPG), that can effectively disinfect bacteria-contaminated water >50 times the mass of the organic content material used. MAPG possess broad-spectrum antimicrobial activity, capable of inactivating >99% of both Gram-negative and Gram-positive bacteria including Escherichia coli, Vibrio cholerae and (methicillin-resistant) Staphylococcus aureus within 20 minutes of treatment. In addition, MAPG can be synthesized in a facile manner from affordable commercially available chemicals. Furthermore, we also demonstrate that MAPG can be fabricated as antibacterial syringes that can be recycled multiple times without losing potency. To the best of our knowledge, MAPG is one of the most potent antibacterial polymer gels ever developed that can potentially be translated to real world water disinfection applications. The excellent activity of MAPG owes to the combination of key two factors: i) the use of facially amphiphilic cationic polymers, and ii) the macroporous structure of the material. While the polymers are responsible for effecting bactericidal property by disrupting the bacteria membrane cell wall in the same vein as antimicrobial peptides and synthetic polymer mimics, the macroporous structure enables the gel to act like a sponge and adsorb the bacteria from the solution, thereby allowing for maximal contact between bacteria and polymer because of the high surface area within the gel internal structure. The platform technology developed herein is not only restricted to water disinfection but may also be applicable in other bacteria inactivation applications.

Results
Material Fabrication and Characterization. To make MAPG, a quaternary ammonium (QA) methacrylate monomer that bears a hydrophobic n-hexyl tail was first synthesized (Fig. 1a). The monomer was chosen on the basis of our experience in developing synthetic antimicrobial polymers, where the judicious combination of cationic and hydrophobic groups is necessary to exert good antimicrobial activity 20,21 . Specifically, the n-hexyl group was selected as sufficient hydrophobicity is required to cause membrane disruption while the ammonium center enables for binding with the anionic character of the bacteria cell wall. The synthesis of the QA monomer via quaternization reaction between 2-(dimethylamino) ethyl methacrylate and 1-bromohexane proceeded in a straightforward fashion and can be performed easily at multigram scale. 1 H and 13 C NMR analysis confirmed successful monomer synthesis (Fig. S1, Supporting Information (SI)). To fabricate MAPG, the QA monomer was polymerized via free radical polymerization in the presence of a redox radical initiator (i.e., ammonium persulfate), coinitiator N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA) and a cross-linkable monomer (i.e., oligoethylene glycol dimethacrylate (OEG-DMA)) in water at subzero temperature (Fig. 1a). The macroporous structure of the gel is generated based on the formation of ice crystals which act as sacrificial templates within the aqueous polymerizable media at subzero temperature. This method of forming macroporous gels is called cryogelation [22][23][24][25] . The overall synthesis process (i.e., from monomer to gel generation) negates the involvement of elaborate lab setup or technique and requires only simple mixing of chemicals, hence making the whole process potentially viable for industrial scale preparation.
The formed MAPG is white in appearance and SEM analysis of the swollen wet gel confirmed the presence of a macroporous structure where the pore size is ca. 15-30 µm in diameter ( Fig. 1b-d). Interestingly, MAPG has buoyancy in water which is due to the porous structure of the material. Compared to an analogous hydrogel (HG), which was prepared at room temperature and hence has no ice crystal templates to yield macroporous structure like MAPG, HG has no buoyancy in water (Fig. 1e). SEM analysis of the wet HG confirmed the non-porous structure of the gel (Fig. 1f). Furthermore, HG appears as a transluscent gel, unlike MAPG which is white and non-transparent. The difference in appearance between MAPG and HG is typical of common cryogels and hydrogels even though they consist of the same chemical composition 22,23 . In terms of swelling ability, MAPG has a higher water uptake than HG where the swelling degree (i.e., the weight ratio of a fully swollen gel to dried gel) for MAPG and HG is 9.5 and 4.5, respectively (Table 1). We also observed that the swelling rate of MAPG is quicker than HG. MAPG achieved a fully swollen state in ca. 10 seconds whereas HG required 500 seconds to be fully swollen (Fig. S2, SI). The superior swelling performance of MAPG is attributed to the porous structure of the material which enables the gel to absorb/uptake large amount of liquid.
The mechanical property of the swollen gels was subsequently determined via uniaxial compression tests. The stress-strain curve as shown in Fig. 1g indicated that both MAPG and HG have similar profile although HG suffered irreversible mechanical fracture when compressed to 19.5% strain of its initial length (at a maximum compression force of 160 ± 8 kPa) whereas MAPG can withstand compression of up to 24.5% strain (compression force of 280 ± 9 kPa). The Young's modulus (E) of MAPG and HG are 567 ± 74 kPa and 683 ± 42 kPa, respectively (Table 1). In general, MAPG and HG which were made from QA and OEG-DMA monomers are mechanically tougher than other reported cryogels and hydrogels where the E values of these materials are typically in the range of 5-30 kPa 15,19 . Besides the covalently cross-linked networks afforded by the polymerization process which keep the polymers interconnected in a gel, we postulate that secondary hydrophobic interactions are present which endow the gels with higher mechanical toughness. Specifically, we believe that the hydrophobic n-hexyl groups from the QA monomer form non-covalent hydrophobic inclusions which further strengthen the gel network, akin to a double network hydrogel system 26,27 . This postulation was supported by the observation that gels made from QA monomer bearing a methyl group, which is far less hydrophobic than the n-hexyl group, are very unstable (easily breakable) and brittle compared to those made from n-hexyl-functionalized QA monomer.

Broad-Spectrum Bacterial Disinfection of Contaminated Bulk Water.
The antimicrobial activity of MAPG was assessed against different Gram-negative and Gram-positive bacteria, namely Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae, and Staphylococcus aureus. For this, the fully swollen gels were sliced into thin discs (ca. 15 × 2 mm and 200 mg in weight) and each placed in a well of a 24-well plate. Bacteria-contaminated water (1 mL of ca. 10 8 colony forming units (CFU) mL −1 ) was added to the well and incubated for a predetermined time period (i.e., 20 and 60 min). The amount of viable bacteria that remained in the bulk water after treatment, denoted as 'treated' water, was determined via standard CFU analysis and compared to the untreated sample (i.e., the negative control). It is noteworthy that these bacteria (e.g., E. coli and V. cholerae) were selected on the basis that they are some of the most common bacteria found in contaminated water supply 28 . In addition, we have also included methicillin-resistant S. aureus (MRSA) 29 in our study to ascertain the ability of MAPG to combat antibiotic-resistant strains.    The number of viable bacteria reduced in the bulk water after treatment with MAPG for 20 and 60 min is summarized in Fig. 2 and Table 1. After 20 min of treatment, >2.0 log 10 reduction in CFU was observed for all the bacteria tested, which translates to >99% of viable bacteria removed from the bulk water. Longer incubation time of 60 min resulted in greater reduction in CFU. For S. aureus, no viable bacteria were detected in the bulk water (i.e., >8.2 log 10 reduction in CFU) whereas for Gram-negative bacteria, >4.0 log 10 reduction in CFU was achieved after 60 min of treatment. MAPG was also effective against clinical MRSA strain, removing >99.9% of the live pathogen from the bulk water after 60 min of treatment. The antimicrobial performance of MAPG is extremely efficient and impressive in terms of the amount of bulk water it can disinfect, the broad-spectrum activity, the log 10 reduction in CFU and the treatment time, as compared to other antimicrobial polymer gels in literature 16,17,19 . In fact, the reported antimicrobial polymer gels are only effective in disinfecting small volumes of water absorbed by the gel, not the bulk water, which limits their potential applicability to biomedical devices.
The antimicrobial performance of MAPG was also compared to HG where slices of HG were subjected to the same antimicrobial test in a 24-well plate system using P. aeruginosa and S. aureus as model bacteria. CFU analysis of the bulk water after treatment with HG for 60 min showed only 37.5% and 96.8% reduction of live P. aeruginosa and S. aureus, respectively (Fig. S3, SI and Table 1). The activity of HG is significantly poorer than MAPG. This is not surprising as the porous structure of MAPG provides a high surface area for efficient bacteria adsorption which is not available in HG. This clearly demonstrated the advantage of macroporous gels over traditional hydrogels for antimicrobial applications.
An additional experiment was also performed to determine if MAPG is still feasible in a larger scale operation. For this, a 6-well plate system was used where the size of each well is larger than the 24-well plate. The weight of MAPG and the volume of bacteria-contaminated water used for this experiment were scaled up accordingly. The experiment was performed against E. coli (ca. 10 8 CFU mL −1 ). CFU analysis revealed that MAPG is still very effective when scaled up and resulted in ca. 3.0 log 10 and 4.0 log 10 reductions after 20 and 60 min of treatment, respectively, even though the effectiveness of the 6-well plate system is lower than the 24-well plate system by ca. 2.0 log 10 reduction in CFU (Fig. S4, SI).
Recyclable Antibacterial Syringes. Given the excellent antibacterial properties of free-standing MAPG in a well-plate system, we next tested the efficacy of MAPG in a syringe format similar to a column chromatography  (Fig. 3a). The synthesis of MAPG in the syringe proceeded in the same fashion as before. For testing the antimicrobial performance of MAPG syringes, E. coli was used as the model bacteria. Like in the earlier experiments, the bacteria concentration was kept at ca. 10 8 CFU mL −1 . The bacteria solution was percolated over the syringe at a flowrate of ca. 1 mL min −1 , and the treated water that exited the syringe was subjected to CFU analysis. Visual inspection clearly showed that the treated water is clear (Fig. 3b). In fact, no viable bacteria were detected in the treated water by CFU analysis, which indicated >8.0 log 10 reduction in bacteria counts compared to the untreated controls (Fig. 3c). The recyclability of the syringe was investigated by subjecting it to multiple passes of E. coli-contaminated water for a total of 8 cycles. Remarkably, no viable bacteria were detected in all percolated, treated water samples while visual inspection of the samples showed the waters were clear (Fig. 3c and Fig. S5, SI). In total, the MAPG syringe disinfected 24 mL of bacteria-contaminated water, which is >120 times the mass of the organic material used, with no detectable viable bacteria in all collected fractions. The syringe system outperformed the well-plate system. This is most likely because the contaminated water is forced to fully interact with the gel via a unidirectional movement in the syringe system, as opposed to the well-plate system where contact between the bacteria and the gel is purely based on random Brownian motion. In essence, the contact between bacteria and gel is not as ordered and efficient in the well-plate system than in the syringe system. Nonetheless, it is worth emphasizing that the antimicrobial activity of the well-plate system is still excellent. The results from both the well-plate and syringe systems thus strongly indicate that good contact between bacteria and gel is vital for high antimicrobial performance.

Antimicrobial Mechanism Investigation. Our initial hypothesis on the antimicrobial mechanism of
MAPG is that the bacteria are first adsorbed onto the material surface, followed by subsequent inactivation by the polymers (Fig. 4a). The overall mechanism therefore procedes via contact active mode. In the abovementioned results, the CFU analysis data of the bulk water that has been treated in the well-plate and syringe systems can only confirm the amount of viable bacteria that were still present in the treated water samples, but do not reveal the fate of those that have been adsorbed by the gel. If the proposed mechanism is correct, the majority of the bacteria adsorbed by the gel should be dead. To prove this, a sample of MAPG that has been used in the disinfection of E. coli-contaminated water in a well-plate system was crushed with a tissue-grinder apparatus (Fig. 4b,c), and the contents were analyzed by CFU analysis to determine the viability of the bacteria that were adsorbed by the gel. This method of determining the bacteria cell viability is comparable to standard procedures of determining the bacteria content in tissue samples for in vivo experiments. It is noteworthy that the bulk water was removed prior to the crushing of MAPG. No viable bacteria were detected in the crushed gel aqueous sample, which indicated that all the bacteria that were adsorbed by the gel were dead. This verifies the proposed mode of action of MAPG (Fig. 4d). An additional experiment was also performed to demonstrate that the antimicrobial activity of MAPG was not due to the leaching of polymers into solution which then inactivate the bacteria. Using a well-plate system, MAPG was incubated in fresh phosphate buffered saline (PBS) solution under identical conditions to an antimicrobial susceptibility assay, and the solution was then mixed with E. coli-contaminated water to determine if the solution contained any leached polymers which may be responsible for the perceived antimicrobial properties. CFU analysis revealed there was no significant difference between the untreated E. coli-contaminated water with the ones that have been mixed with MAPG-incubated solutions. This confirmed that the antimicrobial activity of the gel is indeed exerted by the bulk material itself.

Discussion
The development of polymer gels and scaffolds with inherent antimicrobial properties is an emerging field as these materials offer new avenues in combating (multidrug-resistant) bacteria. In this study, we demonstrated the fabrication of a novel material termed macroporous antimicrobial polymeric gel (MAPG) that exhibits excellent inherent antimicrobial activity against a range of Gram-negative and Gram-positive bacteria as a proof-of-concept for point-of-use water disinfection application. The ability to disinfect a wide range of bacteria family is an important criteria for water disinfection technology as water systems contain mixture of microbes. From a synthetic point of view, MAPG is completely organic and is easily produced at subzero temperature via free radical polymerization of monomers and initiators which can be bought commercially or made in a facile manner at multigram scale. This suggests that the material production process could be implemented by industry.
We also demonstrated that MAPG can disinfect bacteria-contaminated water (>50 times the mass of the organic material used) and effectively inactivates >99% of bacteria from the bulk water within 20 min of treatment. Furthermore, we showed that MAPG can also be formulated into a syringe which can be reused multiple times without losing potency. The MAPG syringe can disinfect bacteria-contaminated water that is >120 times the mass of polymer used with an inactivation efficiency of >8.0 log 10 reduction in CFU compared to the untreated controls. The overall antimicrobial performance of MAPG is truly impressive compared to literature reported materials (especially bearing in mind that a high bacteria concentration was applied, ca. 10 8 CFU mL −1 ), and we showed that this is due to the combination of specific amphiphilic cationic polymers used and the macroporous structure of the gel. The capabilities to disinfect water with high bacteria loading and to reuse multiple times are essential for a device to be applied in a practical sense. Although the antimicrobial performance of MAPG is truly remarkable, additional studies are needed in the future to further ascertain the limit of (next-generation) MAPG. In addition, the organic matter content in bacteria-contaminated water needs to be considered for potential real world applications given that MAPG contains hydrophobic groups which may bind with these organic compounds. Another interesting aspect of MAPG is that this technology could potentially be extended to other fields, for instance, as wound healing materials. The macroporous structure of MAPG can potentially be used to load specific biomolecules such as growth factors to be delivered to wound sites whilst the MAPG scaffold itself eliminates any bacteria infection -all in all accelerating the healing process. We envisage that the platform technology of MAPG will find real world applications in point-of-use water disinfection as well as in other related bacteria inactivation applications.

Methods
Materials. 1 The purity of the monomer was confirmed by NMR spectroscopy (Fig. S1, SI).
Gel Fabrication. QA monomer (1.7 mmol, 550 mg) and OEG-DMA (0.17 mmol, 93.3 mg) were prepared in a 7 mL glass vial followed by the addition of Milli-Q water (1.5 g) for dissolution of the contents. Then, PMDETA (68 μmol, 14.1 μL) was added to the solution. The redox initiator APS (34 μmol, 7.7 mg) was added last and the solution was mixed thoroughly. The glass vial was subsequently sealed. The freshly prepared polymerizable media was immediately frozen in liquid N 2 and kept in a freezer at −20 °C for 3 days to obtain MAPG. To obtain HG, the solution was left to stand at room temperature for 3 h. The glass vial that housed the gel was gently broken to recover the gel. The gels were purified by immersing in Milli-Q water (100 mL) for 10 h which included periodic changing of the solvent every 2 h. To obtain MAPG in a syringe, the polymerizable media (ca. 2 g) was prepared in a 6 mL plastic luer-lock sterile syringe and immediately frozen in liquid N 2 and kept in a freezer at −20 °C for 3 days. The gel in the syringe was purified in the same manner as above.
Mechanical Testing. The mechanical properties of the swollen gel samples (10 mm thickness, and diameter ~9 mm) were characterized using Zwick Roell Z-10-UTM at 25 °C. The load was adjusted to 5 kN and a strain rate of 5 mm min −1 was used, and the sample was compressed up to 40% strain of its initial length. Experiments were conducted in triplicate. The experimental parameters are listed in Table S1, SI.
Swelling Degree Studies. The swelling properties were determined by gravimetric analysis. For the determination of the swelling profile, the gel samples were lyophilized until a constant weight. Dried gels were allowed to swell in excess of water and the water uptake by the gels was determined by measuring the cumulative mass increase at a predetermined time interval. It is worthwhile noting that excess surface water was gently wiped off with tissue paper before measuring the mass of the swollen gel. The degree of swelling at time t was calculated as a ratio of M t /M 0 where M t and M 0 are the masses of the swollen gel at time t and dried gel, respectively. The experiment was done in triplicate.
Bacteria Cell Culture. In all assays, glycerol stock of Pseudomonas aeruginosa PAO1, Escherichia coli K12, Vibrio cholerae SIO, Staphylococcus aureus SA31, or methicillin-resistant S. aureus SA60 was streaked onto Luria-Bertani (LB) medium agar plate and cultured overnight at 37 °C. Afterwards, a single colony of bacteria was picked and inoculated in 10 mL of LB medium at 37 °C with shaking at 180 rpm overnight. The overnight culture was diluted 1:100 in 10 mL of LB and allowed to grow to mid log phase at 37 °C with shaking at 180 rpm for ca. 2.5-3 h, then further diluted 1:5 in phosphate buffered saline (PBS) media (pH 7.4) for subsequent antimicrobial assays. The bacteria concentration in the PBS solution was ca. 10 8 CFU mL −1 .
Antimicrobial Assay for Well-Plate System. Fully swollen gels were cut into thin slices of ca. 15 × 2 mm (ca. 200 mg) and each placed into a well of a 24-well plate. Noteworthy, the mass of the organic content per slice (excluding the absorbed water) was ca. 20 mg for MAPG. To each well was added 1 mL of the prepared bacteria solution in PBS, and the plate was incubated at 37 °C with shaking at 180 rpm for 20 or 60 min. After treatment, bacteria cell viability analysis of the treated bulk water was determined by a drop plate method. The bulk water was serially diluted (10-fold) in sterile PBS and plated onto LB agar plate. Colonies were counted and CFU analysis was determined after the plate was incubated for 24 h at 37 °C. All assays included two replicates and were repeated in at least three independent experiments.
SCIENTIfIC REPORTS | (2018) 8:7965 | DOI:10.1038/s41598-018-26202-0 Antimicrobial Assay for Syringe System. With the syringe system, 3 mL of the prepared bacteria solution in PBS was added onto the top of the MAPG syringe. The liquid was plunged through the syringe manually at a flowrate of ca. 1 mL min −1 , and the water that exited the syringe was analyzed by CFU analysis as abovementioned. The MAPG syringe was recycled consecutively for a total of 8 times using fresh bacteria solution. The breakthrough volume is ca. 240 mL g −1 of polymer material. This assay included two replicates and was repeated in three independent experiments. Antimicrobial Mechanism Investigation. A slice of the MAPG gel, which was used in the 60 min treatment of E. coli-contaminated water in a well plate system, was washed twice with PBS and subsequently placed in a 15 mL Dounce tissue grinder (Sigma-Aldrich), followed by the addition of PBS (5 mL). The gel was thoroughly crushed with a pestel until a slurry is formed. The mixture was homogenized in PBS by incubating in an ultrasonication bath (150 W, 40 Hz) at room temperature for 2 min. The mixture was then subjected to the same CFU analysis procedure. This assay was repeated in three independent experiments. Statistical Analysis. One-way classification of ANOVA and student's t-test (two-tailed) were performed where differences between data were regarded as statistically significant with p-values < 0.05.