C. burnetii appears specifically dependent on molecular iron for replication and viability and exhibits a response to iron akin to bacteria that colonize iron-rich environments. Physiological adaptation of C. burnetii to the unique acidic and degradative environment of the CCV is consistent with access of this pathogen to molecular iron.Host organisms restrict the availability of iron to invading pathogens in order to reduce pathogen replication. To counteract the host’s response to infection, bacteria can rely on redundant mechanisms to obtain biologically diverse forms of iron during infection.  Coxiella burnetii, the causative agent of Query (Q) fever in humans, is a highly infectious obligate intracellular bacterium. Following uptake into a host cell, C. burnetii replicates within a phagolysosome-derived compartment referred to as the Coxiella-containing vacuole (CCV). During infection, C. burnetii exhibits tropism for tissues related to iron storage and recycling , suggesting that pathogen physiology is linked to host iron metabolism. Iron has been described to have a limited role in C. burnetii virulence regulation, despite evidence that C. burnetii-infected host cells increase expression of transferrin receptors, thereby suggesting that active iron acquisition by the bacterium occurs upon infection. Through the use of host cell-free culture, C. burnetii was separated from the host cell in order to directly assess the role of different forms of iron in C. burnetii replication and viability, and therefore virulence. Results indicate that C. burnetii tolerates molecular iron over a broad concentration range  and undergoes gross loss of viability upon iron starvation. C. burnetii protein synthesis and energy metabolism, however, occur nearly uninhibited under iron concentrations not permissive to replication. Despite the apparent absence of genes related to acquisition of host-associated iron-containing proteins, C. burnetii replication is supported by hemoglobin, transferrin, and ferritin, likely due to release of iron from such proteins under acidic conditions. Moreover, chelation of host iron pools inhibited pathogen replication during infection of cultured cells.IMPORTANCE Host organisms restrict the availability of iron to invading pathogens in order to reduce pathogen replication. To counteract the host’s response to infection, bacteria can rely on redundant mechanisms to obtain biologically diverse forms of iron during infection. C. burnetii appears specifically dependent on molecular iron for replication and viability and exhibits a response to iron akin to bacteria that colonize iron-rich environments. Physiological adaptation of C. burnetii to the unique acidic and degradative environment of the CCV is consistent with access of this pathogen to molecular iron. Iron is an essential micronutrient often used as a cofactor in enzymes associated with diverse cellular processes, including respiration and DNA biosynthesis , 2. Alth3–Iron availability in mammals is so tightly regulated that many bacterial pathogens have evolved to maintain redundant mechanisms to adeptly scavenge for this vital micronutrient. These mechanisms include bacterial high-affinity iron transporters and siderophores, iron acquisition receptors of host-associated iron-containing proteins , and direct uptake of bound forms of host iron  –10. More–Coxiella burnetii, the causative agent of Query (Q) fever in humans and coxiellosis in livestock , is a highly infectious zoonotic obligate intracellular bacterium (C. burnetii replicates exclusively within a host-derived compartment referred to as the Coxiella-containing vacuole (CCV)   , 17. Thele (CCV) –20 and aed pulp) , 22, sugC. burnetii virulence regulation . In Legionella pneumophila, a close phylogenetic relative of C. burnetii, FeoAB functions in ferrous iron (Fe2+) transport  culture tools. Specifically, we took advantage of the natural chelation properties of citrate in order to assess pathogen responses to iron. Our results indicate that while C. burnetii tolerates a wide concentration range of iron, the bacterium appears to require unsequestered, molecular iron. This observation remained apparent during C. burnetii infection of host cells, as sequestration of host iron pools via 2,2′-bipyridyl directly inhibits C. burnetii replication, indicating that C. burnetii relies on its host’s labile iron pool (LIP).In the present study, we evaluated the requirement and utilization of iron for C. burnetii have been based on the use of C. burnetii-infected host cells , the citrate-based nutrient medium ACCM-2  (ACCM-2−FeSO4)—determined to contain ∼1 μM iron by inductively coupled plasma mass spectroscopy (ICP-MS) , C. burnetii was unable to replicate when the medium was supplemented with 1 μM FeSO4  media supplemented with 5 or 10 μM FeSO4 (a). C. burnetii was grown axenically in ACCM-2 containing different concentrations of FeSO4 or iron chloride (FeCl3) (b). Each bar represents the average from 3 independent experiments; error bars indicate SEM. Download FIG S1, PDF file, 0.07 MB.ICP-MS was used to determine the relative concentrations of iron in APCMCopyright © 2020 Sanchez and Omsland.2020Sanchez and OmslandCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 4, C. burnetii was incubated in ACCM-2−FeSO4 or in medium supplemented with FeSO4 or iron chloride (FeCl3). Following an 8-day incubation, final yields were determined via quantification of genome equivalents (GE). As expected, FeCl3 supported similar C. burnetii yields as FeSO4, eliminating a critical role for the SO42− supplied via FeSO4 in C. burnetii growth  with only a marginal reduction in final yields , was used. The basal buffer for APCM is based in part on the previously described P-25 buffer . C. burnetii growth deficits in APCM containing 7 mM citric acid could be rescued by additional supplementation of FeSO4 . Each point is the average from 3 independent experiments; error bars indicate SEM. C. burnetii was grown axenically in APCM containing different concentrations of FeSO4 (b). Bacterial yields were measured after 8 days via GE. Each bar represents the average from 2 independent experiments; error bars indicate SEM. Download FIG S2, PDF file, 0.07 MB.Copyright © 2020 Sanchez and Omsland.2020Sanchez and OmslandCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. burnetii appears incapable of effectively utilizing iron complexed to citrate.Many pathogenic bacteria employ specific mechanisms to acquire physiologically relevant iron-citrate complexes , 30. To C. burnetii is dependent on >5 μM FeSO4 in ACCM-2 for optimal replication led to the question of whether this was specific to C. burnetii. Therefore, final culture yields of C. burnetii, Escherichia coli, Pseudomonas aeruginosa, and Yersinia pestis cultured in ACCM-2 or ACCM-2−FeSO4 were compared. Inocula for all organisms were 1 × 106 CFU ml−1, and for bacteria other than C. burnetii medium pH was adjusted to 7.4. Apart from C. burnetii, all bacteria tested had the ability to replicate in ACCM-2−FeSO4, but P. aeruginosa and Y. pestis had an ∼25 to 50% reduction in final yields, respectively, compared to complete ACCM-2 , while yields obtained with E. coli were indistinguishable from the positive control  or in the presence of 5% CO2 and O2 (b). Each bar represents the average from 2 to 3 independent experiments; error bars indicate SEM. Download FIG S3, PDF file, 0.07 MB.Copyright © 2020 Sanchez and Omsland.2020Sanchez and OmslandCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. burnetii viability, CFU assays were performed. For these assays, the chemically defined (citrate-based) axenic medium D-ACM . Bacteria cultured in regular ACCM-2 or ACCM-2−FeSO4 were used as the positive or negative controls, respectively. Absorbance was measured at the start of D.S. and on day 8. Compared to the positive control, C. burnetii cultures that underwent downshifts at 24 h were unable to initiate and/or sustain replication, resulting in significantly lower final yields  incorporation into C. burnetii total proteins (9 GE of C. burnetii was incubated in 0.5 ml of regular ACCM-2 (+ control), ACCM-2 at pH 7.0 (− control), ACCM-2 supplemented with 5 μM FeSO4, ACCM-2−FeSO4 (0 μM FeSO4), or ACCM-2 supplemented with 100 μM iron chelator bipyridyl (Bpdl) for 24 h and then transferred to a labeling medium, and the amount of incorporated [35S]Cys-Met was measured after 3 h. Bacteria incubated under positive control conditions had high relative incorporation of [35S]Cys-Met and vice versa under negative control conditions  did not correlate with a reduction in [35S]Cys-Met incorporation   . OverallC. burnetii synthesis of ATP—for example, to establish an energy charge suitable for replication (−FeSO4 (0 μM), ACCM-2 containing 5 μM FeSO4, or ACCM-2 containing 100 μM Bpdl. Conditions were maintained for 48 h before the relative bacterial ATP pools were quantified. Similar to data obtained for protein synthesis, there was no significant difference between control cultures and bacteria incubated with suboptimal levels of iron   . While tC. burnetii genome suggests the bacterium harbors few genes related to iron acquisition, especially for complexed or bound forms of iron. Therefore, we assessed the ability of C. burnetii to utilize sequestered and bound forms of iron during axenic replication. We confirmed that C. burnetii is unable to replicate axenically when iron is sequestered with Bpdl . To determine whether effects contributed by Bpdl could be rescued with additional iron, ACCM-2 cultures containing 100 μM Bpdl were supplemented with additional FeSO4 and C. burnetii final yields were measured via absorbance (b). C. burnetii final yields were measured by absorbance in APCM−FeSO4 containing different concentrations of Bpdl (c). Each bar represents the average from 2 to 3 independent experiments; error bars indicate SEM. The effect of Bpdl on E. coli viability was evaluated in ACCM-2−FeSO4 containing different concentrations of Bpdl for 24 h (d). Viability was determined at the onset and after Bpdl exposure via CFU enumeration on LB agar. Each data point represents the average from 2 independent experiments; error bars indicate SEM. *, P < 0.05; **, P < 0.01 (unpaired Student’s t test). Download FIG S4, PDF file, 0.08 MB.Copyright © 2020 Sanchez and Omsland.2020Sanchez and OmslandCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. burnetii likely encounters iron-containing molecules, including ferritin, hemoglobin, and transferrin. To determine whether C. burnetii has the ability to utilize host-associated bound forms of iron, ACCM-2−FeSO4 was supplemented with various concentrations of transferrin, ferritin, or hemoglobin, and final yields were measured after 8 days via quantification of GE. Optimal C. burnetii yields were supported by ≥0.25 mg ml−1 transferrin and 0.1 to 1 mg ml−1 of hemoglobin without toxic effects  and C. burnetii appears to exhibit preference for molecular iron, we tested whether a moderately acidic pH induces iron release from transferrin, ferritin, and hemoglobin. For these analyses, 0.5 mg ml−1 samples of transferrin, ferritin, or hemoglobin were incubated in ACCM-2 inorganic basal buffer (4) adjusted to pH 7.3  or 4.75  for 2 h at 37°C and then analyzed by ICP-MS to measure iron content. For transferrin and ferritin, the relative concentration of free and/or released iron following incubation increased significantly at pH 4.75 versus 7.3, with a corresponding loss in bound iron  and incubated for 3 days using cell culture media containing 50, 100, and 200 μM Bpdl; incubation without Bpdl added to the medium served as the positive control. C. burnetii replication decreased significantly after 3 days with increasing Bpdl concentrations, compared to non-Bpdl-treated cells  or 3 (d3), and infections were maintained for an additional 3 days, with C. burnetii intracellular viability quantified at onset and end of Bpdl treatment. When ≥50 μM Bpdl was added on d0, C. burnetii d3 intracellular CFUs trended but were not significantly lower than d0 CFUs (data not shown). In comparison, only 200 μM Bpdl added on d3 resulted in an overall lower mean value for final CFUs on d6, but again differences were not statistically significant , as Bpdl has been shown to specifically affect the LIP of eukaryotic cells  and fluorescence (GFP) micrographs of Vero cells infected with GFP-expressing C. burnetii and exposed to different concentrations of Bpdl at 0 (a) and 3 (b) days postinfection. Download FIG S6, PDF file, 0.4 MB.Copyright © 2020 Sanchez and Omsland.2020Sanchez and OmslandCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. burnetii for organs involved in iron storage and recycling, we established an axenic culture model to study the effects of iron on C. burnetii physiology. Use of the citrate-based medium ACCM-2 allowed analysis of C. burnetii responses to iron without use of additional chemical chelators known to also sequester transition metals other than iron with significance in bacterial physiology  known to colonize nutritionally diverse niches, consistent with unique adaptation of C. burnetii to a specific niche characterized by availability of molecular iron. Additionally, optimal pathogen protein and ATP synthesis occurs under iron availability that does not support C. burnetii growth. Finally, active sequestration of iron by addition of the membrane-permeant iron chelator Bpdl reduced C. burnetii replication during infection of cultured cells, suggesting C. burnetii partially relies on the host LIP.Motivated by the tropism of ysiology , 41. We Borrelia burgdorferi  and organs in mammalian hosts associated with iron storage and recycling. Of potential significance, C. burnetii harbors the gene ght (CBU0530), which in Neisseria meningitidis confers resistance to heme iron toxicity .The core metabolic machinery of lication . ATP synburnetii , is anotburnetii . These oburnetii . Therefolus spp. ) are capChlamydiaceae  is encoded  or in Vero and J774A.1 host cells (b). Each bar represents the average from 3 to 6 independent experiments; error bars indicate SEM. Download FIG S7, PDF file, 0.07 MB.Iron oxidation states were determined for ACCM-2 and APCM containing 100 μM FeSOCopyright © 2020 Sanchez and Omsland.2020Sanchez and OmslandCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. burnetii exhibited comparable growth kinetics during axenic culture with specific iron-containing proteins or molecular iron  hav. pestis  and P. aruginosa , use lowed genes  and ther arrests , consist changes . TherefoC. burnetii in supporting optimal replication and viability is consistent with genome sequence analysis suggesting iron uptake occurs via the FeoAB transporter. C. burnetii. The moderately acidic and degradative properties of the CCV likely support active degradation and release of iron from iron-containing proteins in the mammalian body. This is supported by demonstrated release of iron from transferrin  upon degradation of transferrin, Fe3+ is released, reduced by STEAP3 to Fe2+, and then shuttled into the host cell cytoplasm via the transporter DMT1 (2+ then resides in the label iron pool (LIP) or is stored bound to ferritin (Ft). Fe3+ appears to be the predominate iron species in axenic cultures and Vero cells but equally prevalent as Fe2+ in J774A.1 cells  was propagated in ACCM-2 and prepared for long-term storage as described previously , pH 4.75, with the following chemical composition: 49.9 mM KH2PO4, 152 mM KCl, 59.9 mM NaCl, 1.96 mM MgCl2·6H2O, 0.007 mM CaCl2·2H2O, 2.5 mg/ml Casamino Acids, 1.5 mM l-cysteine hydrochloride, 0.1 mg/ml peptone, 1.0 mg/ml methyl-β-cyclodextrin, and 12.5% (vol/vol) RPMI 1640 without l-glutamine, supplemented with GlutaMAX . Liquid cultures were established in T-25, 6-well, or 12-well polystyrene cell culture flasks/plates containing 7, 3, or 1.5 ml of medium, respectively, and inoculated with ∼1 × 106 GE ml−1 bacteria, unless otherwise noted. ACCM-2 supplemented with tryptophan, pH 4.5  (C. burnetii NMII Tn7-CAT-GFP) was used  broth and grown at 37°C with shaking (∼200 rpm) overnight. Starter cultures for Y. pestis KIM6+ were prepared in heart infusion broth (HIB) at 25 to 30°C with shaking (∼200 rpm) overnight.eviously , 27. Culeviously . C. burnx ACCM-2 , 27, chex ACCM-2 , 30, and, pH 4.5 , was usewas used . The pH l-glutamine , supplemented with GlutaMAX  and 5% [vol/vol] heat-inactivated serum complex ) at 37°C and 5% CO2. For infections, host cells were seeded in 12-well plates at a density of 2 × 105 cells per 1.5 ml and maintained in complete RPMI 1640 at 37°C and 5% CO2 for 24 h. Subsequently, host cells were infected with 1 × 107 GE ml−1 of bacteria in 1 ml of plain RPMI 1640 . Infections were facilitated by centrifugation at 400 × g for 30 min at room temperature (RT). Following infection, inocula were removed and host cells were washed once with plain RPMI 1640 to remove noninternalized bacteria. Infected cells were maintained in 1.5 ml of RPMI 1640  containing 2% (vol/vol) hi-FetalPlex and incubated at 37°C and 5% CO2.Mouse macrophage-like (J774A.1) cells  and African green monkey kidney (Vero) cells  were maintained in complete RPMI 1640 medium . Bacteria were then resuspended in 0.5 ml of citrate labeling buffer that contained the same concentration of iron sulfate to which the cells were exposed during the preincubation. To allow incorporation of radiolabel into proteins, 10 μl EasyTag Express protein labeling mix  was added directly to each cell suspension, and cells were allowed to incubate for 3 h at 37°C and 5% CO2. Following incubation, cells were washed with 0.2 ml of sodium phosphate saline  and then lysed with 50 μl lysis buffer  and heated to 95 to 100°C for 10 min. Equal volumes of each sample were analyzed via scintillation counting to determine counts per minute (cpm). Use of radioactive materials was approved by the Radiation Safety Officer, Washington State University.Radiolabeling of eviously . Briefly6 GE ml−1C. burnetii, allowed to replicate for 3 days, and then subcultured into medium containing 1 μM FeSO4, 5 μM FeSO4, or 100 μM Bpdl. Original cultures and subcultures were allowed to progress for an additional 48 h under 5% CO2 and 5% O2. At time of sampling, cultures were transferred to prechilled centrifuge tubes and incubated on ice for 3 to 5 min. Bacterial cells were then pelleted via centrifugation . Supernatants were removed, and cells were washed with 1.0 ml of ACCM-2 inorganic basal buffer , and ATP was extracted by heating samples to 95 to 100°C for 5 min. Samples were then transferred to −80°C until analysis. To analyze ATP pools, samples were diluted in 0.09 ml sodium phosphate saline , and 0.01 ml of this mixture was added to 0.09 ml of kit master mix for analysis following the manufacturer’s instructions. The concentration of ATP was extrapolated from a standard curve and normalized to GE determined at time of sampling.Quantification of bacterial ATP pools was performed using an ATP determination kit . Briefly, ACCM-2 cultures were inoculated with ∼1 × 10l buffer  before b−1 holo-hemoglobin, ferritin, and transferrin were dissolved in ACCM-2 inorganic basal buffer  to separate iron bound to intact proteins from the free iron. To process, 0.1 ml concentrated HNO3 was added to the filtrates to extract free iron overnight at room temperature, heated to 80°C for 1 h, and then diluted to a final volume of 5 ml with double-distilled water (ddH2O). Iron content was measured by inductively coupled plasma mass spectroscopy (ICP-MS) using an Agilent 7700 series ICP-MS instrument .To measure pH-dependent release of iron from iron-binding proteins, 1-ml aliquots of 0.5 mg mll buffer  without C. burnetii intracellular replication, 0, 50, 100, and 200 μM of the iron chelator Bpdl was added to the culture medium in duplicate wells, infections were allowed to progress for 3 days, and C. burnetii yields were enumerated via quantification of GE. Representative cultures were imaged via fluorescence microscopy using a Leica Dmi8 inverted microscope . To measure the influence of host iron pools on C. burnetii intracellular viability, infections were subjected to 0, 50, 100, and 200 μM Bpdl on day 0 or 3 postinfection in duplicate and infections were allowed to progress for an additional 3 days before viability was enumerated via a CFU assay. Briefly, the medium was removed and 0.25 ml of Trypsin-EDTA  was added to detach cells. An 0.75-ml amount of ACCM-2−FeSO4, pH 4.75, was then added to the trypsinized host cells and homogenized well using a micropipette before being transferred to a Lysis Matrix H tube . Host cells were subjected to mechanical lysis  via two 20-s pulses at 6.0 m s−1, and then host debris was pelleted by centrifugation for 10 min at 1,500 rpm. Supernatants were serially diluted, and 10 μl of each dilution was spotted on top solid ACCM-2 supplemented with tryptophan, pH 4.5 , bovine transferrin , and equine spleen ferritin  in MΩH2O. To extract iron, 0.1 ml concentrated HNO3 was added to each sample and incubated at room temperature overnight. Samples were then heated to 80°C for 1 h before being diluted to 5 ml in ddH2O, generating final concentrations of 1, 5, and 10 μM FeSO4 for ACCM-2 and APCM, and 10 and 250 μg ml−1 for each iron-binding protein. Iron content was measured as described above by ICP-MS.ACCM-2 containing 5, 25, or 50 μM FeSO4 was added to T-25 flasks in duplicate and incubated under microaerobic conditions . At 0 and 3 days postincubation, 50 μl of medium was added to a 96-well plate in triplicate and analyzed for iron oxidation state using the iron assay kit . The assay was performed following manufacturer’s instructions. To measure iron oxidation states in host cells, cells were incubated to confluence in 10 T-75 flasks. Vero cells were detached from flasks using Trypsin-EDTA , and J774A.1 cells were detached via scraping. Host cells were washed twice with phosphate-buffered saline , resuspended in 1 ml of assay buffer, and transferred to Lysis Matrix H tubes . Host cells were lysed via two 20-s pulses at 6.0 m s−1 before insoluble host debris was removed by centrifugation for 10 min at 16,000 × g, 4°C. The resulting supernatant was used directly. Host cell numbers were determined via direct cell count prior to lysis for normalization purposes.To measure the oxidation state of iron in axenic media, 7 ml of freshly prepared ACCM-2 and APCM containing 100 μM FeSO10H8N2; Sigma-Aldrich, St. Louis, MO)   was dissC. burnetii NMII cultures was added to a 1.5-ml screw-cap tube containing 0.1-mm zirconia beads  and subjected to mechanical lysis  via three 30-s pulses at 5.0 m s−1. Samples were serially diluted, and GE were quantified via detection of the C. burnetii gene CBU1206  was performed as described previously . Briefly CBU1206  using a


NNMT methylates nicotinamide to N1-methylnicotidamide (MNA-1) using S-adenosyl methionine. The rs694539 NNMT polymorphism is associated with non-alcoholic steatohepatitis, and rs1941404 is associated with hyperlipidemia. The rs1421085 FTO is related to poor eating behaviors, and rs3751723 IRX3 is associated with obesity. To investigate the association of rs694539 and rs1941404 NNMT, rs140285 FTO and rs3751723 IRX3 polymorphisms with MNA-1 concentrations, resting energy expenditure (REE) and BMI, we included clinically healthy Mexican subjects 30 to 50 years old, 100 subjects (35 men/65 women) with BMI > 30 kg/m2 and 100 subjects (32 men/68 women) with BMI < 25 kg/m2. Glucose, lipid profile, insulin, leptin, acylated ghrelin, and MNA-1 (LC–MS) were quantified. Resting energy expenditure (REE) was estimated using indirect calorimetry with a Fitmate instrument. Genotyping was performed using PCR–RFLP, and allelic discrimination was examined using TaqMan probes. MNA-1 concentrations and REE were significantly higher in obese subjects. Subjects with the rs694539AA NNMT genotype (recessive model) had lower weight, BMI, and REE. BMI showed an association with HDL-C, triglycerides, MNA-1, acetylated ghrelin, leptin, insulin concentrations, HOMA-IR, REE, and rs1421085. Subjects with the TC or CC genotypes of rs1421085 FTO showed 6 kg and 2 units of BMI more than did those with the TT wild type. The CG of the rs1421085 and rs3751723 haplotypes was associated with BMI. These findings showed that BMI was strongly associated with REE, rs1421085 FTO and the CG rs1421085 FTO and rs3751723 IRX3 haplotypes. We used the GMDR approach in obesity phenotype to show the interaction of four SNPs and metabolic variables.The enzyme nicotidamide-N-methyltransferase (NNMT) regulates adipose tissue energy expenditure through increasing nicotinamide adenosine dinucleotide (NAD Seven of ten adults are obese or overweight  in Mexico2. A persistent state of positive energy balance when food intake consistently exceeds energy expenditure (REE) leads to weight gain and eventually to obesity onset3. Several determinants of 24-h REE, such as body fat-free mass, fat mass, age, gender, ethnicity, glucose tolerance, and familial relationship, reflect an underlying genetic background6. The nicotidamide-N-methyltransferase (NNMT) enzyme is a novel regulator of adiposity and energy expenditure, and it directly alters with NAD+ and SAM, which affects histone methylation, polyamine flux and SIRT1 signaling7. Levels of the NNMT enzyme are high in adipose tissue and liver and low in other organs8. NNMT-knockdown mice show an increased loss of body fat, as indicated by increased energy expenditure and oxygen consumption7. NNMT inhibition by N1-methylnicotidamide, which is the product of the NNMT reaction, increases oxygen consumption in adipocytes7. The expression of NNMT in white adipose tissue is approximately two-fold higher in type 2 diabetes mellitus patients (T2DM) than it is in healthy controls9, with elevated levels of MNA-1 observed in the circulation10 and urine11. The MNA-1 plasma levels correlate positively with BMI11 and inversely with insulin sensitivity9.Obesity has become a global epidemic leading to increased cardiovascular disease, type 2 diabetes, cancer, and others associated pathologiesNNMT gene located on 11q23.1, which harbors several single nucleotide polymorphisms (SNPs). Two recently identified SNPs, rs694539 and rs1941404, were associated with chronic diseases such as hyperhomocysteinemia12, nonalcoholic steatohepatitis13, congenital heart disease14, bipolar disorder15, and epilepsy16. The rs1941404 was associated significantly with hyperlipidemia. The effect of rs1941404 variations on REE may be a primary physiological mechanism that leads to hyperlipidemia17. However, the associations of the rs694539 and rs1941404 variations with obesity and resting energy expenditure were not examined.The NNMT enzyme is encoded by the FTO are strongly associated with human obesity, and carriers of the risk alleles show evidence of increased appetite and food intake19. A recent investigation showed that the rs1421085 risk allele of the FTO gene disrupted ARID5B (AT-rich interactive domain-containing protein 5B) repressor binding, which led to derepression of IRX3 (Iroquois homeobox protein 3) and IRX5 expression during early adipocyte differentiation, triggering alterations in browning20. The risk alleles of the FTO rs1421085 polymorphisms are associated with poor eating behaviors , a higher intake of high fat foods and refined starches with depressive symptoms21. The obesity-associated SNPs in the first intron of FTO are functionally connected with the IRX3 gene. These obesity-associated SNPs directly interact with promoters of FTO and IRX3 in humans22. IRX3-deficient mice have a 25–30% lower body weight, which suggests a role in the increase in basal metabolic rate23.Sequence variants in the first intron of NNMT gene, rs140285 FTO and rs3751723 IRX3 polymorphisms with serum MNA-1 concentrations, REE, and BMI in lean and obese subjects.The present study investigated the association of rs694539 and rs1941404 of the p < 0.00001).The anthropometric and metabolic characteristics of the study groups are shown in Table NNMT, the AA genotype frequency was significantly higher in lean subjects, but no differences in allelic frequencies were found , serum leptin (p = 0.05), and MNA-1 (p = 0.06) concentration  vs. 37.4 (16–52), p = 0.05, and 9.9 (7.6–15) vs. 14.9 (8–20), p = 0.07; respectively).Subjects with TC or CC genotypes of rs1421085 FTO (Table p = 0.066). This last association was first observed in the comparison of BMI for rs1941404 genotypes under the recessive model (p = 0.06). No other SNPs were associated with BMI under the dominant model. The REE was also strongly associated with BMI (p < 0.00001), leptin (p < 0.001), insulin concentration (p < 0.001), and insulin resistance (p < 0.001). No association of the rs694539 NNMT, rs1941404 NNMT, rs1421085 FTO or rs3751723 IRX3 polymorphisms with REE were observed under the dominant model.The association of BMI and REE with metabolic variables and SNPs was evaluated using multiple regression analysis including glucose, HDL-C, triglycerides, MNA-1, acylated ghrelin, leptin, insulin concentrations, HOMA-IR, REE or BMI, and testing each one of the rs694539, rs1941404, rs1421085 and rs3751723 SNPs (Table p < 0.05) and acylated ghrelin (p < 0.001) and positively associated with leptin (p < 0.00001) and REE (p < 0.00001). With the univariate fit, BMI was associated with triglycerides (p < 0.001) and insulin (p < 0.00001) concentrations, insulin resistance (p < 0.00001) and CG rs1421085 and rs3751723 haplotype (p = 0.01) after adjusting for gender and age.Using the same regression model and including the rs1421085 and rs3751723 haplotype, BMI was negatively associated with HDL-C , we tested three logistic regression models, two models that included the four SNPs with and without the other predictors , and a third model with only FTO rs1421085 and IRX3 rs3751723 with the predictors. The models with the other predictors showed better accuracy and gene–gene and gene-environmental interactions , and thermogenesis20. Therefore, an increased IRX3 and IRX5 expression in rs1421085 risk allele carriers, favors a greater amount of white adipose tissue and a consequently higher BMI. This finding is observed in double CC risk allele carriers of this SNP who have a 1.7-fold higher obesity risk compared to the homozygous TT genotype20. In accordance with these data, we found that subjects with one or two risk allele (TC or CC) of rs1421085 FTO had 6 kg and 2 BMI units more than did subjects with the TT genotype. Genetic variants of the FTO and IRX3 genes are in high linkage disequilibrium and associated with obesity. We found the TG haplotype of rs1421085 was more frequently associated with obesity in Mexican subjects. Our work did not find an association of these SNPs with REE, but the editing of rs1421085 in adipocytes from a person with the risk allele restored IRX3 and IRX5 repression, activated the browning expression program and restored thermogenesis20. The rs9939609 FTO was associated with BMI in several reports in Mexican populations39, but rs1421085 FTO was reported in only one study. The present study is the first work that has analyzed the associations of BMI and REE with the genetic variant in the NNMT gene, similar to the association of the rs1421085 FTO and rs3751723 IRX3 haplotypes. We also reported the interaction of four SNPs and metabolic variables with the obesity phenotype in a Mexican population.The obesity-associated SNPs in the first intron of FTO polymorphism and the rs1421085 FTO and rs3751723 IRX3 haplotypes were associated with BMI but not with REE. Using the GMDR approach, we found that the obesity phenotype adjusted to a multifactorial model, with the interaction of the four SNPs and metabolic variables.In conclusion, REE and MNA-1 concentrations were significantly higher in obese subjects. BMI was strongly associated with REE, negatively associated with HDL-C, acylated ghrelin, and positively associated with leptin, and MNA-1 concentrations. The univariate fit showed a positive association of BMI with triglycerides, insulin, and HOMA-IR. The REE was strongly associated with age, BMI, insulin, and insulin resistance. Under the recessive model, subjects with the rs694539AA genotype had lower weight, BMI and REE, and subjects with the rs1941404GG genotype showed higher BMI, leptin, and MNA-1 concentrations than subjects with the other genotypes. The rs1421085 2 and 100 subjects (35%/65% men/women) with BMI > 30 kg/m2.We recruited 200 clinically healthy subjects 30 to 50 years old: 100 subjects (32%/68% men/women) with a BMI between 18.5–25 kg/mWeight was measured using a Roman type Tanita BC533 scale. Height was measured using a SECA 406 Stadiometer to calculate the BMI. Systolic and diastolic blood pressures were measured in a sitting position after a 10-min rest. All measurements were performed in duplicate.2 production) was measured for 15 min. The first 10 min was discarded for a better steady state.REE was evaluated via indirect calorimetry (IC) using the Fitmate device . The instrument was calibrated before each assessment, following the manufacturer’s specifications. For evaluation, the participants were instructed to avoid physical exercise and the consumption of coffee or black tea for 24 h before examination. The measurements were performed in a quiet room with a controlled temperature between 22-24ºC after a 12-h overnight fast, low light and no noise. The participants remained awake and lying in a supine position throughout the evaluation. Oxygen consumption . Insulin, leptin and acylated ghrelin concentrations were measured using an ELISA kit  with a sensitivity of 0.399 µIU/mL (range 3.0–200 µIU/mL) for insulin, a sensitivity of 0.42 ng/mL  for leptin, and 5 pg/mL (range 2.0–250 pg/mL) for acylated ghrelin. Insulin resistance (IR) was calculated using the homeostatic model assessment (HOMA)N1-methylnicotinamide (MNA-1) was measured using liquid chromatography coupled to mass spectrometry (LC–MS) using an electrospray ionization–single quadrupole mass spectrometer . Briefly, samples were deproteinized with trichloroacetic acid (TCA). Chromatographic separation was performed using a reverse-phase XBridge BEH C18 (particle size 2.5 μm) 100 mm × 3.0 mm column (Waters), protected with a C18 (ODS) 4(L) × 3.0(D) mm guard cartridge (Waters) and maintained at 25ºC. All buffers were filtered through a 0.2 μm filter (Phenomenex) under reduced pressure prior to use. Mobile phase consisted of a 10-mM ammonium formate solution tritiated to pH 2.2 by formic acid. The UHPLC operation was performed in isocratic mode with a constant flow rate of the mobile phase at 0.3 mL/min. ESI ionization was performed in the positive ion mode. The mass spectrometer was operated in selected ion recording (SIR). The single ion mode was used with 137.15 m/z for N1–methylnicotinamide. The quantification was performed using the peak area ratio.The plasma 13. The digestion PCR products were loaded onto a 3% agarose gel at 80 V for 90 min and visualized using ultraviolet light transillumination . A band of 187 pb corresponded to the G allele, and two bands of 106 pb and 81 pb were observed for the A allele (13). For genotyping of the FTO rs1421085 polymorphism, we used the primers 5´-TAGTAGCAGTTCAGGTCCTAAGGCGTG-3´(forward) and 5′-CAGATTAAGGTGATGGGTTG-3′ (reverse) at a concentration of 10 pmol, 50 ng DNA, 0.2 mM dNTP´S, 2.0 mM magnesium chloride (MgCl2), Taq polymerase 2 units and buffer Taq 1 × using the amplification program previously described21. A total of 10% of samples were re-analyzed to check the reliability of genotyping, and a 99% matching was obtained.Genomic DNA was isolated from whole blood using a commercial kit. The rs694539 genotyping was performed using PCR–RFLP and the following primers: 5′-AAGTGCTGACAGGTGATAGG-3 '(forward) and 5′-CATCTTTTCACTCTCCTTGC-3′ (reverse) at an aligning temperature of 62 °C for 30 s for 35 cycles. The 187 pb PCR product was digested with the NIaIII restriction enzyme at 37ºC overnightIRX3 rs3751723 and NNMT rs1941404 was performed using a validated Taqman® allelic discrimination protocol (Applied Biosystems®). For the IRX3 (VIC/FAM) G/T transversion substitution,Genotyping of [G/T]GGCTCCGGGCTCTGATTGACATTTC. For the NNMT (VIC/FAM) A/G transition,CCGAACAGATTGGCGGAGATTCCCG[A/G]TGTTAGTAAATTTGTGTATGTGCAC, using TaqMan™ Genotyping Master Mix enzyme . The reactions were performed in a CFX96 Touch  using a 96-plate thermal cycler, according to the manufacturer's protocols. A total of 10% of samples were re-analyzed to check the reliability of genotyping, and 99% matching was obtained.AGATGAGATAGGCCCATGTGTGTGCU test. We compared allelic and genotypic frequencies between groups using χ2 with Yates correction. Associations of BMI and REE with metabolic, hormonal and SNPs variables were evaluated with a multiple regression analysis, and relationships between variables were analyzed used the Spearman correlation coefficient. Analyses were performed using the statistical package Statistica 7 .Anthropometric and metabolic data are expressed as the means ± SD or medians (25–75 quartiles). The differences between lean and obese subjects, such as the comparison between genotypes under the recessive model, were evaluated using Student’s t-test or Mann–Whitney www.goldenhelix.com). The four DNA variants were analyzed for deviation from the Hardy–Weinberg equilibrium using Fisher's exact test. P < 0.01 indicated a statistically significant difference. For the association study, we used a stepwise linear regression model  with recoded genotypes with the additive genetic model . BMI and REE were used as response variables. Multiple Bonferroni and false discovery rate corrections indicated a statistically significant difference. To analyze possible interactions between FTO-IRX3 , we used the generalized multifactor dimensionality reduction (GMDR) approach [https://www.ssg.uab.edu/gmdr/). A p-value < 0.05 was considered statistically significant.Analyses were performed using SNP & Variation Suite v8.8.1  approved this study. All participants signed written informed consent to participate in the study. The study was performed in accordance with the ethical standards of the Declaration of Helsinki in 1983 and in agreement with the Good Clinical Practice guidelines.Supplementary file1 (XLSX 42 kb)


AIF, which efficiently transported cargos into epithelial cells was exanimated. CPPAIF was evaluated with cell model and 3D skin model following OECD guidelines without using animal models. As a highly stable peptide, CPPAIF neither irritated nor sensitized skin, also did not disrupt skin barrier. In addition, such high safety peptide had anti-inflammation activity without allergic effect. Moreover, cargo carrying activity of CPPAIF was assayed using HaCaT cell model and rapid CPPAIF penetration was observed within 30 min. Finally, CPPAIF possessed transepidermal activity in water in oil formulation without disruption of skin barrier. All evidences indicated that CPPAIF was an ideal choice for skin penetrating and its anti-inflammatory activity could improve skin condition, which made CPPAIF suitable and attractive for novel cosmeceutical product development.Cosmeceutical peptides have become an important topic in recent decades in both academic and industrial fields. Many natural or synthetic peptides with different biological functions including anti-ageing, anti-oxidation, anti-infection and anti-pigmentation have been developed and commercialized. Current cosmeceutical peptides have already satisfied most market demand, remaining: “cargos carrying skin penetrating peptide with high safety” still an un-met need. To this aim, a cell-penetrating peptide, CPP Peptides, polymers composed of amino acids, are known to possess versatile biological functions such as promoting cell proliferation, migration, inflammation/anti-inflammation, angiogenesis, and melanogenesis , which cCosmeceutical peptides usually have certain features. Historically, it has always been assumed that the molecular weight of a peptide should be less than 500 Da, if not it could not pass skin barrier . In addiScientific research on peptides usually focus on identification of functional mechanism, and practical application for cosmetic and/or pharmaceutical use. In the last decade, the development of active peptides has established a new field in cosmeceutical and pharmaceutical skin care. To generate safety profile and functional data of peptide, in vivo animal test was usually chosen in the past. However, correctness of results from animal model for human skin has always been quested. This problem, using animals for testing purposes, finally leads to EU regulation , beginning in 2009, to prohibit use of animals to accumulating toxicological data for cosmetic ingredients.® , EpiDerm® , SkinEthic®  and epiCS® . Recently, some advanced skin models were commercialized, including Phenion®  and NeoDerm® . These models have been proven to replace animal tests in pharmaceutical and cosmetic industries for evaluation of corrosion, skin irradiation and photo-toxicity [As an alternative solution, artificial human skin models have been established and many of these are now commercially available. Several technologies have been introduced to design and develop artificial skin models which highly simulate complex structure of human skin ,7. The mtoxicity . Some oftoxicity .Human skin gives protective, perceptive and communication functions to the body with resilient and relatively impermeable barrier. To develop agents to deliver pharmacy or active ingredients across skin tissues is a highly attractive topic in recent years. Using compounds or physical equipment to enhance cargo delivery causes some problems, for instance, skin toxicity, skin irritation, inconvenience and high costs ,11. CompAIF (anti-inflammatory CPP). CPPAIF has been shown to possess epithelial cell, GAG and lipid binding properties as well as cell penetrating activity through macropinocytosis [AIF is able to deliver small fluorescent molecules, recombinant proteins, nanoparticles, and peptidomimetic drugs into cells [AIF for cosmeceutical application were examined with skin cell and 3D-skin models following the Organization for Economic Co-operation and Development (OECD) guidelines with special focus on stability, safety, skin irritation, skin barrier function, chemico sensitization, bio-functions and transepidermal activity in this work.Here a 10-residue peptide, covering major GAG binding motif of a human ribonuclease, is identified as a CPPocytosis ,21. Notato cells . Based oAIF  and was dissolved in water, concentration of 1 mg/mL, and then freeze and dry into powder. Samples were separately incubated in 4 and 25 °C for 1, 3 and 7 days. These tubes were collected and stored at −80 °C. For solution stability test, CPPAIF was dissolved in water to 1 mg/mL then used 0.2 µm filter filted and dispense to 100 µL in each tubes. Samples were separately incubated in specific temperatures, including −20, 4, 30 and 50 °C for 1, 3, 7, 14, 21, 30 and 60 days. These tubes were collected and stored at −80 °C. Then, the remainder of CPPAIF were tested with high-performance liquid chromatography (HPLC)  Separation was performed on XBridge C18 column . The HPLC condition and program: A buffer is ddH2O with 0.1% TFA (trifluoroacetic acid), B buffer is acetonitrile with 0.1% TFA. The flow rate is 1 mL/min and acetonitrile gradient from 10 to 50% in 15 min and the percentage of acetonitrile raise to 100% from 16 to 20 min.All chemicals used in this study were purchased from Sigma-Aldrich . All cell lines were purchased from ATCC . For dry powder stability test, CPPAIF cause skin irritation [AIF) to surface of epidermis and subsequent assessment of effect on cell viability. All 3D-skin tissues were incubated with growth medium for 2 h and then CPPAIF was add to final 1 mM for 42 min treatment. After treatment, testing substance was washed out by 25 times with 1 mL phosphate buffered saline (PBS) and tissues were further incubated in growth medium for 42 h. After incubation, growth medium was substituted by maintenance medium with 3--2,5-diphenyltetrazolium bromide (MTT) agent for 3 h incubation. Next, insert (with tissue) were washed with PBS and air dried. Formazan in tissues was extracted with isopropanol and measured by determining the OD at 570 nm using a microplate spectrophotometer . Cell viability of treated models was normalized to the negative control, which was set to 100%.The 3D reconstructed human epidermis tissue model: SkinEthic™ RHE  was used to evaluate whether CPPritation . TestingAIF on the barrier function on SkinEthic™ reconstructed human epidermis (RHE) based on the relevant procedure mentioned in OECD TG 439. This testing procedure involved topical application of CPPAIF onto surface of the epidermis model for 1 h. After 1 h of exposure, CPPAIF was washed by PBS from the surface followed by application of detergent solution (1% Triton X-100) onto surface of the tissue for another 2 h. Then, washed the detergent solution and AlamarBlue cell viability assay  was performed for assessment of cell viability. Cell viability of treated models was normalized to the negative control, which was set to 100%.A test procedure was developed to test effect of CPPin chemico method which quantified the remaining concentration of cysteine- or lysine-containing peptide after 24 h incubation with the test chemical at 25 °C. Relative peptide concentration was measured by HPLC  with gradient elution and UV detection (220 nm). Cysteine and lysine peptide percent depletion values were calculated for a prediction model (The direct peptide reactivity assay (DPRA) is an on model . This moAIF displayed anti-inflammation potential [5 cells/mL) and allowed to attach overnight. After attachment, cells were incubated with various concentrations of CPPAIF for 1 h and followed by stimulation with 1 μg/mL of LPS (lipopolysaccharide). No LPS-added cells were considered as control groups. After incubation, the amount of TNF-α and IL-6 in the medium were analyzed by enzyme linked immunosorbent assay . Cell viability was measured by AlamarBlue cell viability assay .Macrophage inflammatory assay were carried out to evaluate whether CPPotential ,23,24. I5 cells/mL RBL-2H3 cells were seed in 24-well plates  containing 10% Fetal Bovine Serum (FBS), streptomycin (100 µg/mL) and penicillin (100 units/mL)) and sensitized with anti-DNP IgE (0.45 µg/mL). These cells were washed with Siraganian buffer , and 40 mM NaOH, pH 7.2) with glucose, 1 mM CaCl2, and 0.1% bovine serum albumin (BSA). After washing, these cell were incubated in 160 µL of the PBS for 10 min at 37 °C and then 20 µL of test sample solution or calcimycin  was added and further incubated for 10 min. The plate was cooling on ice for 10 min to stop reaction. 50 µL of supernatant was transferred to 96-well plate and added with 50 µL of substrate (1 mM p-nitrophenyl N-acetyl-β-d-glucosaminide) in 0.1 M citrate buffer (pH 4.5) at 37 °C for 2.5 h. The reaction was stopped by adding 200 µL of carbonate-bicarbonate buffer . The value of A405 was measured with a microplate reader.Inhibitory effects on release of β-hexosaminidase in RBL-2H3 (rat-basophilic leukemia cell line) were evaluated by a cell degranulation assay . BrieflyAIF  in human keratinocytes, HaCaT. First, apply a layer of type I collagen on a glass cover slip, dry and sterilize the cover slip with UV light. Then, culture the HaCaT cells (1 × 103 per slip) on the coverslip for 16 h. The attached HaCaT cells were cultured with 20 μM of the TMR- CPPAIF for 30 min, observed with CLSM (63× oil-immersion objective lens), and photographed under same exposure conditions.Confocal Laser-scanning Microscopy  was performed to assess distribution of TMR-labeled CPPAIF-containing emulsion systems (water-in-oil emulsion (W/O) and oil-in-water emulsion (O/W)) were prepared for the transepidermal measurements. Formulation contents of the W/O and O/W are listed in AIF (0.1 mM) emulsions were add to top of the tissues for 1 h treatment. After treatment liquid beneath the model was collected for HPLC quantification. Transepidermal rate was calculated by measuring the amount of CPPAIF in medium beneath the 3D model by HPLC  × 100).3D human epidermis tissue model  and two kinds of CPPAll statistical analyses were processed by GraphPad Prism version 5.01 for Windows 548 . Each value was the average of three measurements, where the presented data was the mean ± SD and all means were compared by one-way ANOVA.AIF was incubated at a specified temperature (4 °C and 25 °C) and analyzed remaining quantity by HPLC. The molecular weight of CPPAIF was validated by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry. The result indicated that m/z of CPPAIF was 1381.1 as expected (AIF maintained intact close to 100% in dry powder form at low temperature (4 °C) and normal temperature (25 °C) up to 1 week, indicating that preservation of CPPAIF in dry powder form could effectively prevent peptide precipitation and fragmentation.CPPexpected . The resAIF under different solutions or temperatures was further examined to explore suitable storage condition. It was dissolved in ddH2O at different temperatures and time to examine which solution was suitable for storage. First, CPPAIF was dissolved in water at a concentration of 1 mg/mL passed through 0.2 µm filter, and incubated at different temperatures. After one week, residual quantity of CPPAIF was still more than 90% when tested at −20 °C and 4 °C, but that of CPPAIF incubated at 30 °C and 50 °C dropped to about 70%   . MALDI-Tmatogram .AIF were carried out following OECD Test Guideline No. 439 (2015), using a reconstructed human epidermis test method.In vitro skin irritation testz of CPPAIF (1 mM) was determined to be respectively: 100.0 ± 4.8%, 1.44 ± 0.8%, and 90.3 ± 3.2%, clearly suggesting that CPPAIF (1 mM) did not cause skin irritation (no category) according to the classification of OECD 439.As shown in AIF was subsequently applied to in vitro skin barrier function test (developed from OECD TG 439). A normal stratum corneum was multilayered containing essential lipid profile to produce desired functional barrier with robustness to resist rapid penetration of cytotoxic chemicals, e.g., SDS or Triton X-100. Here, a 3D human epidermis tissue model, SkinEthic™ RHE, was used for skin barrier function tests. Tissues incubated with CPPAIF (1 mM) remained cell viability over 80%, which was considered not influencing barrier function of the tissue. As shown in 2O), positive control , and CPPAIF (1 mM) was respectively 100.0 ± 6.2%, 24.0 ± 3.3%, and 95.6 ± 2.8%, evidently indicating that CPPAIF would not impair the skin barrier function of the 3D epidermis tissue model.CPPAIF was tested following OECD Test Guideline No. 442C. The mean of cysteine and lysine % depletion of 100 mM cinnamaldehyde (positive control), phenoxyethanol, caprylyl glycol, hexalene glycol, 1,3-butanediol and 0.1 mM CPPAIF was respectively calculated to be 65.07, 0.56, 2.53, 0.63, −0.12 and 0.74  and the amount of TNF-α or IL-6 inhibition. Nevertheless, cell viability remained 85.2 ± 4.25% at CPPAIF concentration of 1 μM. Taken together, CPPAIF might be a potential ingredient with skin protectant function, especially anti-photo aging [Inflammation inhibition effect of CPPectively B. These to aging ,27 relatto aging .AIF was evaluated by mast cell degranulation assay. Here RBL-2H3 cells were treated with various concentrations  of CPPAIF. Granule release represented by β-hexoaminidase was induced by A23187  as positive control (PC). In this experiment CPPAIF under 10 μM did not induce any mast cell degranulation release . Nuclei were stained with DAPI . As shown in AIF  for 30 min, TMR signal was clearly detected in the cells  * 100). As shown in AIF), CPPAIF present at the bottom of the 3D skin tissues W/O and O/W formulations was determined to be respectively 41.4 ± 3.6% and 28.4 ± 1.7% respectively in W/O and O/W formulations. The transepidermal activity of W/O CPPAIF was evidently higher than that of O/W formulation. Taken together, appropriate formulation such as W/O emulsion in this study could evidently facilitate transepidermal penetration of CPPAIF.Transepidermal degree of CPPAIF. As shown in AIF penetration into stratum corneum while at the same time maintained the robustness barrier function of the epidermis. The 3D models incubated with W/O CPPAIF showed a higher relative cell viability of 76.5 ± 8.5% than O/W CPPAIF formulation (43.3 ± 7.8%). Relative cell viability of negative control  equaled to 100.0 ± 9.6%. Thus, W/O CPPAIF displayed a better transepidermal degree with slight disruption effect on disruption of skin barrier function.We further examined skin barrier function of the 3D model after 4 h of exposure to formulated CPPSince 2000 the application of peptides in cosmeceutical products has rapidly increased, and this trend has sped up research and knowledge of physiological properties of peptides. Now, researchers have identified peptide sequences for penetration into skin layers or different cosmetic activities  [AIF was tested with regulatory affair approved methods and clear formulations, therefore the safety datum and anti-inflammatory activity were highly credible. CPPAIF in W/O or O/W formulation could pass through 3D human tissue model. Our CPPAIF showed skin penetration activity without disrupting skin barrier function, and its anti-inflammatory activity might alleviate slight inflammation caused by conventional transepidermal methods [AIF was convinced to be a perfect choice for carrying active ingredients through skin tissue and that will be our next goal.Here CPP methods . As a CP methods . This st methods . Unlike  methods , hence i methods . To reduAIF, a GAG binding peptide, could penetrate cell membranes with cargos in living animals and was proven to be stable in powder form under room temperature or in water solution under −20 °C using HPLC analysis. Following OECD guidelines, CPPAIF was evaluated and characterized without skin irritation, skin sensitization and did not disrupt skin barrier function using 3D skin model. In addition to high safety CPPAIF was identified to inhibit inflammation by decreasing inflammatory cytokines, TNF-α and IL-6, using macrophage model. With this bio-function, CPPAIF was further proven to not have sensitization effects. The result of penetration test in HaCaT cells verified CPPAIF for cargo (TMR in this case) delivery into cell in less 30 min. Finally, two commonly used formulations were applied to evaluate transepidermal activity of CPPAIF in 3D skin model in order to imitate real cosmetic applications, and W/O formulation of CPPAIF was identified as a better choice to efficiently penetrate 3D skin with slight disruption of skin barrier function.CPP


However, the molecular mechanism for the effects of Leu on muscle growth and protein deposition is not clearly demonstrated in fish. This study investigated the effects of dietary Leu on growth performance and muscle growth, protein synthesis, and degradation-related signaling pathways of hybrid catfish (Pelteobagrus vachelli♀ × Leiocassis longirostris♂). (2) Methods: A total of 630 hybrid catfish (23.19 ± 0.20 g) were fed 6 different experimental diets containing graded levels of Leu at 10.0 (control), 15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 g Leu kg-1 for 8 weeks. (3) Results: Results showed that dietary Leu increased percent weight gain (PWG), specific growth rate (SGR), FI (feed intake), feed efficiency (FE), protein efficiency ratio (PER), muscle fibers diameter, and muscle fibers density; up-regulated insulin-like growth factor I (IGF-I), insulin-like growth factor I receptor (IGF-IR), proliferating cell nuclear antigen (PCNA), myogenic regulation factors , and MyHC mRNA levels; increased muscle protein synthesis via regulating the AKT/TOR signaling pathway; and attenuated protein degradation via regulating the AKT/FOXO3a signaling pathway. (4) Conclusions: These results suggest that Leu has potential role to improve muscle growth and protein deposition in fish, which might be due to the regulation of IGF mRNA expression, muscle growth related gene, and protein synthesis and degradation-related signaling pathways. Based on the broken-line model, the Leu requirement of hybrid catfish (23.19-54.55 g) for PWG was estimated to be 28.10 g kg-1 of the diet (73.04 g kg-1 of dietary protein). These results will improve our understanding of the mechanisms responsible for muscle growth and protein deposition effects of Leu in fish.(1) Background:  The majfor fish . Leu defa crocea , rainbows mykiss , juvenils ovatus , grass c idellus , black cn piceus , fingerlunctatus , juvenileolatus♂ , juvenilycephala , Catla cla catla , and stifossilis . Neverthfossilis ,25. Averfossilis . These dThe process involved in the increase in muscle growth is associated with accumulation of protein ,28. The Pelteobagrus vachelli♀ × Leiocassis longirostris♂ is a hybrid catfish that has been widely cultured in China in recent years. Our previous studies have determined optimal dietary tryptophan levels [n levels . Therefo−1 and crude fat 71.0 g kg−1. Different concentrations of Leu were added to a basal diet mixture to constitute the seven levels of 10.0 (control group), 15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 g Leu kg−1. A mixture of crystalline l-amino acids was supplemented to simulate the muscle essential amino acid pattern of hybrid catfish except for Leu  with a 2 mm die. The processing conditions were as follows: 100 rpm screw speed, 127 °C temperature, and 30–45 atm pressure. Floating extruded pellets were air-dried and stored at 4 °C in plastic bags until use.Leu was obtained from Heng Yuan Biotech. Co. . The composition of the experimental diets is given in  for Leu . All die3), resulting in 30 fish in each tank. Fish were fed with their respective diets to apparent satiation two times  per day for 8 weeks at a feeding rate of about 3%–5% their weight. Average water temperature was 25 ± 2 °C. Dissolved oxygen was higher than 5 mg L−1 and pH was maintained at 7.0 ± 0.3. Continuous water flow was maintained at the rate of 1.2 L·min−1 in each tank. Fish were kept under a natural light and dark cycle. All experimental protocols were approved by the Animal Care Advisory Committee of Sichuan Agricultural University.Hybrid catfish were purchased from Rong Sen Corporation . Fish were adapted to the experimental environment for one month. A total of 630 fish with an average initial weight of 23.19 ± 0.20 g were randomly distributed into 21 tanks (200 × 100 × 105 cm−1). After slaughtering, fish were immediately filleted using a new sterile scalpel. Muscle samples from the left side of 6 fish in each tank were quickly frozen in liquid nitrogen and then stored at −80 °C until the determination of RNA content, gene expression, and Western blot. Muscle samples from the right side of the same fish were obtained for histological analysis.After a fasting period of 24 h, all fish in each tank were weighed and counted at the initiation and termination of the feeding trail. Prior to sampling, 6 fish from each tank were anaesthetized in benzocaine bath  following the manufacturer's protocol. Total RNA quantity and quality were assessed by electrophoresis on 1% agarose gels and spectrophotometry at 260 and 280 nm. The 2 μL total RNA was used to synthesize cDNA using the PrimeScript® reverse transcription kit with gDNA Eraser (TaKaRa). Specific primers were designed according to the sequences cloned in our laboratory and the published sequences of hybrid catfish . All of The processes for muscle protein extract preparation and Western blotting were conducted according to the procedures as previously described . BrieflyP < 0.05 was considered to be statistically significant. Pearson correlation coefficient analysis was conducted using the Bivariate Correlation program. Dietary Leu requirement of hybrid catfish were estimated by the broken-line model.The results were represented as the means ± standard error of mean (SEM). The data were subjected to one-way analysis of variance, which was followed by Tukey’s method, to determine the significant differences among the groups by SPSS 20.0 . −1 (P < 0.05), and no significant differences were found among other groups. The percent weight gain (PWG), specific growth rate (SGR), and feed efficiency (FE) were gradually increased for fish fed diets with increasing Leu levels up to 25 g kg−1, then gradually decreased (P < 0.05). The feed intake (FI) was the highest for fish fed the 25 g Leu kg−1 diet, and lowest for fish fed the 40 g Leu kg−1 diet (P < 0.05). The PER was highest for fish fed the 25 g Leu kg−1 diet, and lowest for fish fed the control diet (P < 0.05). Based on the broken-line model, the dietary Leu requirement of hybrid catfish for PWG was estimated to be 28.10 g kg−1 of the diet, corresponding to 73.04 g kg−1 of dietary protein . The muscle fiber density was higher for fish fed Leu-supplemented diets than control diet (P < 0.05). The protein content .The microstructure of white muscle in cross-section is shown in  content A and RNA content B gradual−1, and decreased thereafter (P < 0.05). The effects of dietary Leu on muscle growth related gene mRNA expressions are presented in −1 had higher mRNA level of Myf5 in muscle than those fish fed other diet groups (P < 0.05). The MyoD, MyoG, and PCNA mRNA expressions gradually increased with increasing Leu levels up to 20 g kg−1, and decreased thereafter (P < 0.05). In comparison with the control group, muscle mRNA level of MRF4 was higher in fish fed 20.0 and 25.0 g Leu kg−1 diets (P < 0.05). Fish fed 25 and 30 g Leu kg−1 diets had a lower level of MSTN mRNA expression than those fish fed the control diet (P < 0.05). The MyHC mRNA level was the highest for fish fed 30 g Leu kg−1 diet, followed by 20, 25, and 35 g Leu kg−1 diets, then control and 15 g Leu kg−1 diets, and the lowest for fish fed the 40 g Leu kg−1 diet (P < 0.05).As shown in P < 0.05). No significant difference was observed in the AKT mRNA level among different dietary Leu levels. The TOR mRNA level in muscle was the highest for fish fed the 20 g Leu kg−1 diet, followed by the 25 g Leu kg−1 diet, then the 15 g Leu kg−1 diet, and the lowest for fish fed the control diet (P < 0.05). Fish fed 20, 25, and 30 g Leu kg−1 diets had lower levels of 4E-BP1 mRNA than those fish fed the control diet (P < 0.05). Fish fed diets containing 25.0 g Leu kg−1 had higher mRNA levels of S6K1 in muscle than those fish fed the control diet (P < 0.05).The effects of dietary Leu levels on protein synthesis related signaling molecule mRNA levels in hybrid catfish are presented in −1 and 40 g Leu kg−1 diets than control diet (P < 0.05). The P-AKT, P-TOR, and P-S6K1 protein levels were higher for fish fed the 20 g Leu kg−1 diet compared with those fish fed 10 and 40 g Leu kg−1 diets (P < 0.05). Fish fed the 20 g Leu kg−1 diet had higher ratio of P-AKT and AKT (P < 0.05).The effects of dietary Leu levels on the AKT/TOR signaling pathway are presented in  and AKT C, P-TOR  and AKT D, and P- and AKT E than th−1 diet, and the highest for fish fed the 40 g Leu kg−1 diet (P < 0.05). The mRNA levels of MURF-1 and MAFBX gradually decreased for fish fed diets with increasing Leu levels up to 20 g kg−1, and increased thereafter (P < 0.05).The effects of dietary Leu levels on protein degradation related signaling molecule mRNA levels are presented in −1 diet compared with those fish fed 10 and 40 g Leu kg−1 diets (P < 0.05). The P-FOXO3a protein level was higher for fish fed the 20 g Leu kg−1 diet than those fish fed 10 and 40 g Leu kg-1 diets (P < 0.05). Fish fed the 20 g Leu kg−1 diet had higher a ratio of P-FOXO3a and FOXO3a than those fish fed the control diet (P < 0.05). However, no significant difference in the MURF-1 protein level among treatments was detected (P > 0.05).The effects of dietary Leu levels on protein degradation related signaling molecule protein expressions are presented in −1 of the diet, corresponding to 73.04 g kg−1 of dietary protein. This value (g kg−1 of dietary protein) was similar to that of large yellow croaker (67.90 g kg−1 of dietary protein) [−1 of dietary protein) [−1 of dietary protein) [−1 of dietary protein) [−1 of dietary protein) [−1 of dietary protein) [−1 of dietary protein) [−1 of dietary protein) [Catla catla (47.90 g kg−1 of dietary protein) [−1 of dietary protein) [−1 diet resulted in a significant reduction in growth. Similarly, excess Leu in the diet also appeared to have adverse effects on growth of other fish species [Salvelinus namaycush [As an essential amino acid, dietary Leu level had a clear effect on growth and feed utilization of hybrid catfish. The optimal dietary Leu requirement for maximal PWG of hybrid catfish was estimated to be 28.10 g kgprotein) , lower tprotein)  and juveprotein) , but higprotein) , black cprotein) , fingerlprotein) , juvenilprotein) , juvenilprotein) , Catla cprotein) , and stiprotein) . The wid species ,56,57, a species . A previ species . On the amaycush .P = 0.066, P = 0.019), MyoD , MyoG , and MRF4  mRNA levels  and AKT  mRNA levels (P= 0.014), which suggested that dietary Leu promotes muscle protein synthesis by activating the PI3K/AKT signaling pathway via IGF-I. Similar results were observed in muscle of rainbow trout and fine flounder [P= 0.000) and S6K1  mRNA levels, and negatively correlated with 4E-BP  mRNA level , MURF-1 , and MAFBX  mRNA levels (P = 0.028), MURF-1 , and MAFBX  mRNA levels  for PWG was estimated to be 28.10 g kg