Escherichia coli (MPEC) is related to an acute mastitis and its treatment is still based on the use of antibiotics. In the era of antimicrobial resistance (AMR), bacterial viruses (bacteriophages) present as an efficient treatment or prophylactic option. However, this makes it essential that its genetic structure, stability and interaction with the host immune system be thoroughly characterized. The present study analyzed a novel, broad host-range anti-mastitis agent, the T4virus vB_EcoM-UFV13 in genomic terms, and its activity against a MPEC strain in an experimental E. coli-induced mastitis mouse model. 4,975 Single Nucleotide Polymorphisms (SNPs) were assigned between vB_EcoM-UFV13 and E. coli phage T4 genomes with high impact on coding sequences (CDS) (37.60%) for virion proteins. Phylogenetic trees and genome analysis supported a recent infection mix between vB_EcoM-UFV13 and Shigella phage Shfl2. After a viral stability evaluation (e.g pH and temperature), intramammary administration (MOI 10) resulted in a 10-fold reduction in bacterial load. Furthermore, pro-inflammatory cytokines, such as IL-6 and TNF-α, were observed after viral treatment. This work brings the whole characterization and immune response to vB_EcoM-UFV13, a biocontrol candidate for bovine mastitis.Bovine mastitis remains the main cause of economic losses for dairy farmers. Mammary pathogenic  In terms of the lactation period and specifically regarding clinical mastitis, Escherichia coli, Streptococcus uberis, Streptococcus dysgalactiae and Staphylococcus aureus are the main etiological agents involved that have been routinely isolated5. Among these pathogens, mammary pathogenic Escherichia coli (MPEC) is responsible for an acute mastitis characterized by inflammation, increased somatic cell count (SCC) and impaired milk quality even after the infection has been cured6.Mastitis treatment is typically based on the use of short and long-acting antibiotics, respectively, during the lactation and dry periodE. coli strains obtained from different types of mastitis (e.g per-acute and persistent) and the non-pathogenic strain K71, Blum et al. (2017) performed a mammary immune response comparison in experimentally infected cows and noticed differences regarding TNF-α, IL-6 and IL-17 secretion levels for each MPEC.Using in vitro and in vivo assays13.Within the current scenario of widespread antibiotic-resistant bacteria, bacterial viruses present as an efficient therapeutic or prophylactic tool in order to control different pathogens in dairy cows at different lactation stages. This is supported by current 14. Lytic viruses related to T4 have awoken interest for their application in phage therapy due the absence of lysogenic modules, a broad-host-range (from Proteobacteria to Cyanobacteria phyla) and, recently, the identification of virion-associated peptidoglycan hydrolases (VAPGHs), which are considered potential enzybiotics17.Considered a model system in molecular biology, coliphage T4 has been studied since the 1940s and possesses about 300 genes organized in a 168.9 kb linear dsDNA with an average GC content of 34.5%18 in the first safety test of phage therapy in humans and in immunological assays in order to elucidate the interactions between viruses, its host and the immune system. Bocian et al.19 investigated how purified T4 phage and T4-generated E. coli lysate impact immune cells differentiation, highlighting that lysis of Gram-negative bacteria by phages might not trigger excessive monocyte induction.Indeed, T4 was used by BruttinE. coli and S. aureus26. The use of animal models is time and cost effective approach, along with a previous step for pre-clinical and clinical assays27. However, the absence of intrinsic cow factors can be considered a bottleneck when data is analyzed28.Currently, several studies have been exploited the use of mouse models in the attempt to evaluate novels anti-mastitis drugs mainly against Escherichia phage UFV13, a T4virus, in genomic, protein and physiological terms and evaluate the immune response in an experimental E. coli-induced mastitis mouse model with the aim to use it in clinical trials to control mastitis in dairy cows.The aims of the present study were to characterize the Escherichia coli 30 following the well-established Sambrook & Russell29 protocol. This virus belongs to the bacterial virus collection of the Laboratório de Imunovirologia Molecular (LIMV) at Universidade Federal de Viçosa (UFV), Viçosa city, Brazil.vB_EcoM-UFV13 (UFV13) was obtained from the sewerage system of Viçosa, Minas Gerais state, Brazil and was propagated on −1 was adopted for both columns. Finally, viral titer was measured at 37 °C by double-agar overlay method30 using E. coli 30 as the plating host. Phage stocks were stored at 4 °C for further analysis.After virus propagation, viral particles were purified by a three-step protocol using ion exchange and desalting columns in a chromatography system . Briefly, an initial step using HiTrap Desalting prepacked column  was conducted to remove any salts used at virus propagation stages and the first two peaks were collected and purified using an ion exchange chromatography column, with specific fractions (7 to 11) driven to the final step, a new desalting process. For the anion exchange column, start  and elution buffers  were used, whereas in desalting steps a phosphate buffer  was prepared. Flow rate of 5 mL.minet al.31. Hypothetical proteins were manually checked for homologs using UNIPROT database. Protein isoelectric point and molecular weight were obtained using ExPASy32. Putative tRNAs and Rho-independent transcription terminators were, respectively, predicted using tRNAscan-SE33 and ARNnold web tool34. The Database of Gene Regulation in Bacteriophages (phiSITE)35 was used in order to check the three major classes of promoters  as well as to confirm putative Rho-independent terminators. The CGView Server was used to generate a UFV13 genome graphical map36.Phage genome extraction, sequencing and annotation methodologies are described according to Duarte 37 (parameters: LEADING:10 TRAILING:10 SLIDINGWINDOW:4:15 MINLEN:65) and aligned to Enterobacteria phage T4 using Bowtie2 software (v2.2.4)38. SAMtools39 was used to convert the output SAM format to BAM format and, subsequently, to sort the BAM file. The sorted BAM file was processed with mpileup tool (SAMtools package) in order to extract the variants. The Binary Call Format (BCF) created was converted to VCF format using BCFtools39. VCF file and Enterobacteria phage T4 genes were used as input for SnpEff program40. Only variants with predicted “high” or “moderate” impact on the protein-coding gene were analyzed.Nucleotide differences between UFV13 and Enterobacteria phage T4 (accession number AF158101.6) genomes were performed by checking reading alignments. High-quality Illumina reads were filtered and adaptor sequences were removed using Trimmomatic software (ver 0.33)Tevenvirinae  deposited on the International Committee on Taxonomy of Viruses (ICTV) were downloaded from NCBI (accession numbers are provided in Supplementary Table 41. Whole-genome single nucleotide polymorphisms (SNPs) were extracted as described in Treu et al.42 and a SNP-based phylogenetic tree was constructed using PHYLIP package43 and visualized by dendroscope44. Furthermore, a whole-genome phylogenetic tree was formulated aligning previously cited genomes from the Tevenvirinae subfamily. Online tools ClustalW245 and MAFTTT version 746 were used for multiple alignment. A neighbor-joining tree was drafted with MEGA747 and visualized with FigTree (http://tree.bio.ed.ac.uk/software/figtree/).Whole-genomes that represent each genus from the subfamily In silico bacterial hosts of UFV13 were predicted using HostPhinder48.With the aim of obtaining the UFV13 structural protein profile, proteomic analysis was conducted.g, 15 min), viral pellet was resuspended in 1 mL of SM buffer and one volume of chloroform followed by centrifugation at 4,000 g for 10 min. An equal volume of trichloroacetic acid (TCA) 10% (v/v) was added to the supernatant and incubated on ice for 30 min. The precipitated viral proteins were collected by centrifugation , washed three times with acetone  and resuspended in water. The BCA Protein Assay Kit  was used to estimate protein concentrations.Viral propagation/purification (see item 2.2) were conducted and phage particles concentrated by adding NaCl 1 M and polyethylene glycol 8000 (PEG8000) 10% (w/v). The mixture was kept overnight at 4 °C. After a centrifugation  was acquired by matrix-assisted laser desorption-ionization time of flight (MALDI/TOF-TOF) (Ultraflex III - BRUKER DALTONICS). Spectra intervals between 500 and 3,500 kDa were selected and forwarded for MS/MS analysis. Results were obtained by Mascot™ (Matriz Science) software using the NCBl nr protein database. Only proteins and peptides indicated as significant by Mascot were considered for further analysis.For protein profiling, the viral proteins were separated via sodium dodecyl sulfate-polyacrilamide electrophoresis (SDS-PAGE) on a15% gelet al.51. In each of the following cases, following treatment the phage preparation was diluted and tittered as described above (2.2).Physiological features were assessed in triplicate following the protocols described by Jurczak-Kurek Viral capability at different acidic and alkaline pH values was evaluated. One mL of purified viruses were transferred to 9 mL LB medium with pH 2, pH 4, pH 7 (control), pH 10 and pH 12 at a 1:9 ratio and incubated for 1 h at 37 °C, being proceeded by a 10-fold serial dilution and plating as described in item 2.2. After overnight incubation at 37 °C, virus stability was determined by the percentage of viruses able to produce lysis plate.Thermal stability studies on LB-diluted phage suspensions were made at −20 °C, 40 °C, 62 °C and 95 °C, respectively, for 12 h, 40 min, 40 min and 5 min. After these periods, a serial 10-fold dilution was conducted and a specific aliquot was plated and incubated overnight at 37 °C. Viruses that did not undergo any thermal treatment were used as a control.Viral replication at different temperatures  was assessed by spot-assay after 24 hours of incubation using a 10-fold serially diluted viral stock in LB medium.4; pH 7.2) with sodium chloride 4.5 M, incubated at room temperature for 15 min and quickly diluted in TM buffer without sodium chloride. Bacterial viruses incubated in TM buffer without sodium chloride were used as a control.In order to verify the effect of the osmotic shock on virus particles, a stock aliquot was transferred to TM buffer , sodium lauroyl sarcosinate (Sarkosyl) and cetyltrimethylammonium bromide (CTAB) on virus particles was determined incubating a standardized viral suspension with 0.09% SDS (20 min at 45 °C), 0.1% CTB (1 min at 22 °C) and 0.1% Sarkosyl (10 min at 22 °C). Controls were done considering the same conditions for each antiviral compound but they were substituted for TM buffer.4; pH 7.2) were prepared and used for plating. Phages incubated in TM buffer under conditions described above, were used as a control.To study the effect of four different organic solvents, a viral suspension was added to 63% ethanol, 90% acetone, 90% chloroform and 50% dimethyl sulfoxide (DMSO). Mixtures were incubated for 1 h at 22 °C (ethanol and acetone), 1.5 h at 4 °C (chloroform) and 10 min at 4 °C (DMSO). In the next step, 10-fold dilutions in TM buffer  Dairy Cattle . E. coli 30 was evaluated for its ability to form biofilm as well as for motility capacity. Motility assays were conducted according to Deziel et al.52. Briefly, an overnight E. coli 30 aliquot was washed, suspended in distilled and sterilized water, and inoculated in King B medium , supplemented with 1.5, 0.5 and 0.3% of agar for twitching, swarming and swimming tests, respectively. Biofilm assay followed the common crystal violet (CV) staining method53. After incubation (37 °C for 48 h), wells were washed 3 times with PBS buffer to remove not adherent cells. CV was added at a minimum volume capable overcome bacterial suspension volume, making possible quantify all bacterial biomass, and incubated for 30 minutes at room temperature. To biomass quantify, CV was removed, wells washed with PBS, added ethanol to solubilize biomass internal CV crystals, and optical density measured at 560 nm. All assays were performed in triplicate.The mammary-pathogenic E. coli 30 was assessed by disc diffusion assay using polisensidiscs for 25 different antibiotics  following the manufacture’s recommendation. The results were interpreted according to the standards of the Clinical and Laboratory Standards Institute (CLSI)54.Antimicrobial susceptibility test of E. coli 30 in vivo, an experimental E. coli-induced mastitis mouse model was used. The trial followed the methodology described by Chandler55, with some modifications, and was approved by the Ethics Committee (Comissão de ética no uso de animais/UFV) according to the protocol 64/2016. Lactating Balb/c female mice (5 to 15 days) was intraperitoneally anesthetized with 10% Ketamine and 2% Xylazine, with subsequent surgical operation of the mammary gland by cutting the teat canal of the last two abdominal ceilings  , PBS and phage served as the control groups, while phage plus bacteria was considered the treatment group (MOI 10). Viral addition was done four hours after bacterial inoculation. Three animals were used to perform each experimental group.With the aim to evaluate UFV13 effectiveness and the immune response against 56 was performed to estimate E. coli 30 colony-forming unit (CFU).In order to assess the lytic activity of the phage vB_EcoM-UFV13 in mammary glands, animals were euthanized 48 hours after the treatment by anesthetic overdose. Infected mammary glands were removed and transferred to 1.5 ml of (PBS), macerated and serially diluted (1:10) in PBS buffer. A microdrop assayCytokines IL-6, TNF-α, IL-2, IFN-γ, IL-4, IL-10 and IL-17A obtained from macerated mammary gland  were simultaneously quantified by the Cytometric Bead Array kit  in a BD FACSVerse Flow cytometry following manufacturer’s recommendations.For histology, mice were euthanized 48 hours after phage treatment. The mammary glands were removed and fixed in Karnovsky fixative . Further, the tissues were embedded in paraffin and a 5 µm section was obtained by microtomy, stained with hematoxylin and eosin (H & E) and observed under light microscopy. The images were acquired under an Olympus DP73 microscope.post hoc test.Statistical analysis was performed with GraphPad Instat 3 software  using the one-way analysis of variance (ANOVA) at 95% accuracy level to evaluate the differences between mammary gland cytokine production under different conditions. This assay was set up in triplicate and, for each animal, the two abdominal ceilings were pooled into one sample. Tukey’s test was used as 57.Bacteriophage UFV13 was isolated from samples obtained in the sewage system of Viçosa, Minas Gerais, Brazil, a well-known source of novel viruses58. In addition, a total of ten tRNA encoding genes  were identified and are organized in a gene cluster without introns or pseudogenes , 269 ORFs were predicted and annotated. The size is similar to that of other members of the T4virus genusI-TevI, I-TevII and I-TevIII are absent, as with some of their related introns like mob and seg genes  identified 10 tRNAs for both pSs-1 and Shfl2 viruses infecting Shigella spp. as host. Despite little existing knowledge about tRNAs functioning in phage life cycles, high tRNAs numbers found in some bacteriophages could be proportional to phage genomes sizes and are related to a short latent period and high burst size value60.Similarity analysis using the deposited genomes from Yersinia phage PST and Shigella phage Shfl2 showed that the bacteriophage UFV13 has 97% identity with both phages. However, it has a larger percentage of aligned genome (96%) for the Shfl2, and 90% of genome aligned to phage PST .In the total, 35, 21 and 31, early, middle and late promoter regions respectively, were predicted, which corresponds to 73% of the promoters identified for the T4 genome  and 30.9(b) were, respectively, detected on the ORFs 52, 163, 178 and 208. According to Miller et al. (2003), generally 34 terminators are found in the T4 genome. The absence of terminators for the genes nrdA and 56/segF can be explained by their absence, since these genes were not annotated. The significance of terminators within genes is unknown but have been described for T4 phages14.Overall, termination of transcription in T4 bacteriophages occurs by an intrinsic termination signal, a stem-loop arrangement accompanied by a U-rich region, or a Rho-dependent termination mechanism62. The large number of SNPs found in virion proteins reflects the diversification of phage UFV13 from the classical T4 and can be associated with the high capability to infect different bacterial genus such as Escherichia, Morganella and Shigella, an uncommon phage feature (data not shown).A total of 5,071 filtered variants  were identified (1 variant every 33 bases) between UFV13 and T4. 83,656 effects were assigned and categorized (395 high 0.472%), 1,670 low (1.996%), 2,722 moderate (3.25%) and 78,869 modifier (94.28%). The number of effects by functional class is: missense 2,879 (60.79%), nonsense 187 (3.95%) and silent 1,670 (35.26%). Considering only genes with high or moderate variants, the mainly affected protein category was that related to virion structural proteins, with gp7 being the most affected , a whole-genome tree was constructed. The phylogenetic tree divided phages into four clusters with vB_EcoM-UFV13 possessing the closest relationship with Shigella Shfl2 and Yersinia phage PST , Yersinia , Shigella  and Salmonella .With the aim to establish the host range of phage UFV13 host range, E. coli chaperonin GroL (57.36 kDa), Escherichia phage vB_EcoM_112 major capsid protein (56.09 KDa) and E. coli outer membrane protein C (36.77 kDa). These proteins are deposited at UNIPROT under accession numbers A0A160CBJ3, A0A017I9Q1, A0A160CBB0 and A0A148HSV3, correspondingly. The presence of a common viral receptor (OmpC) and the chaperonin GroL highlights the need for viral purification enhancement. Viral isoelectric points are usually below six and some of them are able to bind to anion and cation exchange matrixes. However, host cell DNA is efficiently removed by cation exchanger columns, while host cell proteins are effectively eliminated by anion exchange63.SDS PAGE analysis revealed the presence of eight proteins  Fig. . InteresE. coli at 30 °C and 22 °C with a plating efficiency of 69 and 56%, respectively , while viral titer dropped to 50% when UFV13 was incubated for 40 min at 62 °C Fig. . No signIt was found that osmotic pressure change, detergent and organic solvents determined a significant titer drop of UFV13. A survivability of 12% was observed when UFV13 underwent a rapid dilution from high-concentration NaCl buffer to low-concentration ones Fig. . The aniet al. (2016). In a broad physiological study using 83 bacteriophages isolated from urban sewage, the same source of UFV13, it was verified that the vast majority of phages were sensitive to a temperature of 62 °C , were able to survive in basic pH (ranging from 10 to 12), were susceptible to detergents and organic solvents, except chloroform, and were also resistant to osmotic shock.Physiological features evaluated in this study are in accordance with Jurczak-Kurek +/Cl− and pH65. Evaluating a bacteriophage cocktail composed of two T4 phages in raw milk against E. coli, Porter et al. (2016) observed a 3.3- to 5.6-log reduction of bacterial growth over a 12-h physiologic temperature. As discussed below (item 3.4), this study showed UFV13 activity against E. coli in lactating female mice.Viral stability assays have also been shown that UFV13 can survive in raw milk and it has potential capability to survive in mastitic milk. In general, mastitis can dampen the quality of raw milk composition, which includes increased levels of NaE. coli 30, an isolate obtained from a dairy cow with acute mastitis, displays resistance to 14 of 25 evaluated antibiotics  the E. coli 30 was positive for biofilm formation, swarming, and swimming, being negative only for the twitching   were measured, however only IL-10, TNF-α and IL-6 were identified as statistically significant  activated or not by lipopolysaccharide (LPS), Bocian et al. (2016) observed that both preparations considerably increased the percentage of CD14+CD16−CD40+ and CD14+CD16−CD80 + monocytes in LPS-unactivated PBMCs cultures, as well as the concentration of IL-6 and IL-12. Notwithstanding, this result suggests that T4 bacteriophages may act both as a pro-inflammatory inducer, and also as CD40 activator, for this reason the increased expression of IL-6, IL-10, and TNF-α as a consequence of the presence of contaminating LPS left after the purification step rather than a property of the virion.Studying whether T4 bacteriophage and T4-generated In vivo assessment of cytokine patterns followed by a single-dose T4 bacteriophage application in an E. coli induced mastitis mouse model, our work indicates that IL-10 levels decreased locally in phage therapeutic group when compared to the negative control (PBS buffer)  revealed that the maximum concentration of IL-10 occurred after 72 hours and was correlated with a decreased level of TNF-α. This finding is in accordance with our results. Reduced IL-10 levels  did not provoke an additive effect on the production of any pro-inflammatory cytokine. Indeed, the IL-6 level diminished , IFN-γ levels were detected in all groups, however no statistical difference was observed between the groups, which was also applicable to IL-2  Fig. . IL-10 hels Fig.  and increls Fig.  and IL-6els Fig.  is indiched Fig.  after phE. coli induces Th1 T-cell responses. Th1 pattern is involved in a cellular immune response that protects against intracellular infections by viruses and microorganisms that grow in macrophages74.The absence of detectable IL-4 and IL-17A cytokines, respectively present in Th2 and Th17 inflammatory responses, indicates that the use of T4 phage in mammary glands infected with E. coli 30 (~100 cells) were inoculated (Fig. Histological analysis of the mammary gland in the control group Fig.  shows inted Fig. . Inoculated Fig. .Figure 7E. coli dairy cows.Viral genomic analysis is a crucial step in bacteriophage screening for their use as an antibacterial agent. The UFV13 virus has no lysogenic modules or genes conferring antibiotic resistance. Viral stability analysis in the presence of detergents and organic solvents and incubation at different pHs and temperatures, have shown that the UFV13 virus presents high survivability at basic pHs and is relatively resistant under incubation at 62 °C for 40 min. vB_EcoM-UFV13 used in an animal model for mastitis reduced the total bacterial load by 90%, as well as inducing pro-inflammatory cytokines such as IL-6 and TNF-α, which makes it a potential biological agent capable of controlling acute infections caused by Supplementary Information


BHO2 and BHO3), have better anti-giardiasic activity than omeprazole in concentrations around 120–130 µM, without cytotoxic effect on mammal cell cultures. The derivatives inactivated GlTIM through the chemical modification of Cys222 promoting local structural changes in the enzyme. Furthermore, derivatives forms adducts linked to Cys residues through a C-S bond. We demonstrated that PPI can be used as scaffolds to design better antiparasitic molecules; we also are proposing a molecular mechanism of reaction for these novel derivatives.Giardiasis is a worldwide parasitic disease that affects mainly children and immunosuppressed people. Side effects and the emergence of resistance over current used drugs make imperative looking for new antiparasitics through discovering of new biological targets and designing of novel drugs. Recently, it has determined that gastric proton-pump inhibitors (PPI) have anti-giardiasic activity. The glycolytic enzyme, triosephosphate isomerase (GlTIM), is one of its potential targets. Therefore, we employed the scaffold of PPI to design new compounds aimed to increase their antigiardial capacity by inactivating GlTIM. Here we demonstrated that two novel PPI-derivatives ( Giardia lamblia , the causal agent of giardiasis, is a flagellated protozoan parasite that lives in the small intestine. The infection can be asymptomatic or produce abdominal pain, chronic diarrhea2, failure to thrive syndrome, and delays in the physical growth and cognitive-intellectual development of infants4. Giardia is a cosmopolitan parasite, but its prevalence is rising in developing countries; therefore, it is categorized into the neglected diseases group5. Besides, a rising incidence of giardiasis in children of daycare centers was identified, assigned it as a re-emerging infectious disease6. Giardiasis affects in all ages; however, immunosuppressed groups and children between 1–5 years old are mainly affected7. There are 280 million of cases worldwide reported with 500,000 new symptomatic cases every year9. Current drug therapies include the use of benzimidazoles and 5-nitroimidazoles such as tinidazole, secnidazole, being metronidazole the most commonly employed. Nevertheless, there are reports describing drug resistance in strains from animals and human patients, as well as unwanted side effects by using such drugs15. The mentioned above, stand out the requirements of novel drug design therapies against this parasite, along with the discovery of new targets whose structural or functional changes disturb the viability and growth of parasite. It has been described the Giardia glycolysis as a potential biological target to design novel anti-giardiasic compounds17. This metabolic pathway is considered one of the main sources of ATP synthesis for parasite18, since Giardia does not carry out oxidative phosphorylation19. The newly- synthetized Nitro(benzo)thiazole acetamide derivatives give rise to promissory results showing an enhanced in vitro anti-giardiasic activity. One of the potential drug targets is the glycolytic enzyme, fructose-1,6-biphosphate aldolase20. We showed that triosephosphate isomerase of G. lamblia (GlTIM), may be a potential specie-specific drug target. The enzyme is a homodimer that catalyzed the interconversion between D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. We reported elsewhere that the chemical modification (derivatization) of the non-catalytic residue Cys222 by the gastric proton-pump inhibitor omeprazole, induce the total inactivation of GlTIM16. This Cys222 derivatization disturbs the hydrogen bond lattice that affects both the binding of substrate and the catalytic mechanism, similar to observed before with other compounds22. Omeprazole also showed an in vitro anti-giardiasic effect that correlates with the GlTIM inactivation from cells, suggesting an in vivo involvement of this enzyme as target16.in vitro anti-giardiasic ability. To reach these aims, three derivatives named BHO1, BHO2 and BHO3  and BHO3 with the pyridine substituents of lansoprazole (3-methyl-4-trifluroethoxpyridine) , which contain an equivalent Cys residue, the Cys217 ; for BHO2 and BHO3, the rate constant values were respectively 1.6 ± 0.1 and 1.2 ± 0.06 M−1s−1 showing that both analogs are more faster than the proton-pump inhibitor. Thus, the substitutions in both rings, the methoxy by methyl group in benzimidazole and the changes performed in pyridine, substantially improved the inactivating reactivity of analogues against GlTIM.To advance with the inactivating characterization of derivatives, we determined their second-order rate constant values of inactivation and then were compared with that obtained for omeprazole. BHO2 and BHO3; then aliquots were withdrawn in appropriated times in order to obtain around of 75, 50 or 20% of residual activity. Afterwards, we added to aliquots the reducing agent dithiothreitol (DTT) and their residual activities continued monitoring. Figure To shed light about the reaction mechanism of omeprazole and derivatives in GlTIM, we performed experiments guided to determine if after its derivatization, the release of adducts attached to enzyme reverts the inactivation, assuming that binding is mediated by disulfide bridge. Hence, GlTIM was incubated at 37 °C with fixed concentrations of omeprazole, BHO2 and BHO3 treatment was respectively 2.02 ± 0.02, 1.9 ± 0.2 and 2.15 ± 015. This indicates that two Cys residues were modified by treatment of compounds. An aliquot of each derivatized enzyme was withdrawn and incubated with 5 mM DTT during 15 minutes; then, the samples were filtered and the free Cys was again quantified. The number of Cys/monomer was 2.2 ± 0.2, 2,1 ± 0.22 and 1.8 ± 0.18 for omeprazole, BHO2 and BHO3, respectively. Hence, the data showed that, notwithstanding the evaluated compound the number of free Cys per monomer does not change, showing that reducing agent did not be able of release the adduct from Cys residues. Therefore, the data strongly suggest that compounds are not attached to the Cys residues of GlTIM through disulfide bridges.We evaluated the first option by determining the residual free Cys after derivatization with compounds and afterwards reducing agent treatment. Thus, 6 mg/mL (214 µM) of GlTIM was incubated with or without each one of compounds (1 mM) during two hours at 37 °C. It is important to remark that at the protein concentration used for this aim, a considerable quantity was precipitated. Therefore, the incubation of the samples was arrested by centrifuging and then filtration through a 3 mL Sephadex G-25 column, and the number of free Cys quantified from the soluble protein. The GlTIM without treatment showed 3.95 ± 0.2 Cys/monomer; this was in concordance with the theoretical number of expected residues, i.e., 4 Cys/monomer. In contrast, the number of Cys per monomer obtained for omeprazole, Given that structural perturbation might contribute in the mechanism of irreversible inactivation, we evaluated the conformational changes of GlTIM by its derivatization with the compounds. We first investigated changes in the secondary structure by monitoring the circular dichroism (CD) signal of GlTIM modified by compounds and free of treatment; however, no appreciable changes were detected Fig. . BesidesBHO3, whereas close of 17% decreased by omeprazole and BHO2 treatment. Otherwise, the mass center spectra of GlTIM were not affected by compounds treatment . Such a modification was identified in two peptides containing Cys14, two peptides containing Cys127 and one peptide with Cys222. BHO3 treatment also rendered peptides with an adduct equivalent to benzimidazole moiety (Δm 131.06), one peptide containing Cys14 and three with Cys222. However, unexpectedly an adduct with Δm 367.09 was also clearly detected in 23 peptides containing Cys14, indicating that the reaction between this residue and BHO3 render a product with both rings  contains fragmentation spectra of sufficient quality for a reliable assignment. This modification was identified in seven peptides containing Cys14, eight peptides enclosing Cys127 and another three peptides including Cys222 in their sequences. Similarly, for 48.06 conBHO2 induce the loss of pyridine ring. In contrast, BHO3 is capable to form two adducts: the first one preserves both rings of molecule while the second one only conserves the benzimidazole ring like those generated by omeprazole and BHO2.Altogether, the data indicate that compounds modify Cys222, but also the Cys14 and Cys127 are susceptible to derivatization. Besides, the route that follows the linkage of both omeprazole and in vitro. Figure 50 value from each compound was calculated. Thus, for BHO2 and BHO3 the values  were around of the half of that obtained for omeprazole (300 µM). These results indicate that, in parallel with the increased inactivator effect on GlTIM, changes in substituents increase the cytotoxic effect of analogues over the trophozoites.The anti-giardiasic ability of compounds was tested in an 50 values calculated and depicted in Table 50 of 14 mM, this value is 46-fold higher than that obtained for trophozoites cytotoxicity . For human fibroblasts, omeprazole did not show cytotoxic effect. Likewise, BHO2 and BHO3 exhibited CC50 values for mammal cells in the millimolar ranges, which are 38- to 61-fold higher respect to the IC50 obtained for trophozoites; thus, analogues also exhibited low in vitro intrinsic cytotoxicity over eukaryotic cells.The cytotoxic effect of these compounds was also evaluated in mammal cell cultures and their CCG. lamblia, important efforts to search novel targets and design new anti-giardiasic drugs have been performed28. On this line, we and other research groups previously determined that omeprazole has anti-giardiasic activity17. Further, it has been shown that the loss of trophozoites viability correlates with a reduction of endogen GlTIM activity, suggesting its participation as one of the drug targets16. Therefore, we consider to omeprazole as lead molecule that can be take it as scaffold to design better anti-giardiasic drugs. In consonance to this aim, we design three derivatives  to increase their ability to inactivate the GlTIM, along with intensifying the giardicidal effect, but having low cytotoxic activities. The synthesis through two-steps method afforded yields above of 70%, where a later chromatographic step increased their purity (≥99%). Hence, excepting BHO1, the other two compounds improved the inactivation against GlTIM respect to omeprazole. The modest inactivating capability of BHO1 (20%) along with the absence of substituents in its pyridine ring, disclose the essential contribution of these latter on the reaction mechanism. BHO2 changed the methoxy by a methyl group and preserved the original substituents of omeprazole in the pyridine ring; this changes improved its solubility but notably, its reactivity against GlTIM (three-fold). For BHO3, nonetheless this compound had an increment of reactivity (two-fold), the contribution of trifluoroethoxy group in the pyridine ring was more limited. It must be underline that neither BHO2 or BHO3 increased their reactivity against mutant GlTIM C222A, confirming that GlTIM inactivation is promoted by derivatization of Cys222, as have been reported for other compounds22.Owing to the rising of resistant strains of current drugs against 21. From latter studies and this one, it was concluded that at least two particularities may contribute to this specificity. The first is that pockets surrounding the Cys222 and Cys217 are quite different, i.e., from the seven residues forming the pockets only one is conserved in both enzymes. Consequently, the distribution of electric charges of each pocket is dissimilar. Furthermore, the accessible surface area value of the residues surrounding the Cys217 of HsTIM is higher than that in Cys222 from GlTIM21. Second, inactivation effects by C222 provoke perturbations of loop 7, a region whose interrelationship with loop 6 is essential for stability, ligand binding and catalysis of all TIM’s. In contrast, the Cys217 modification of HsTIM by compounds or mutations, did not affect appreciably its catalysis or stability24. This suggest that the pocket of Cys217 does not interact with some one of the mentioned secondary structures.The high specificity of reaction for the Cys222 of GlTIM compared with its equivalent in HsTIM, the Cys217, has been observed with several compounds other that omeprazole23 this suggests that reaction mechanism follows the route reported for proton-pump inactivation with omeprazole30. In contrast, our data show that the compounds bind covalently to Cys residues by non-disulfide links; this data is of relevance to understand the chemical mechanism of reaction. In this regard, Im et al.31 proposed that at neutral pH, the benzimidazole moiety of omeprazole attached to a SH- group of mercaptanes by non-disulfide links. Given that our experiments were performed at neutral pH, it is reasonable to deduce that compounds bind to Cys yielding similar species. The data mentioned before, together with mass spectrometry results allow proposing a reaction mechanism of compounds , forming the adduct “B”  promotes barely structural modifications Fig. ; howeverin vitro studies showed that BHO2 and BHO3 have better anti-antigiardiasic effects than omeprazole, but without increasing their cytotoxicity in eukaryotic cells cultures. This data support our proposition about omeprazole can be used as scaffold to synthesize better anti-giardiasic compounds. Interestingly, albeit BHO2 and BHO3 displayed different inactivation rate constant values for recombinant GlTIM, they exhibited very similar IC50 values for the in vitro assays. These results suggest that besides of GlTIM, the compounds may have another target in G. lamblia. This could be a very interesting property since multi-target activity of drugs increases their anti-parasitic potential because of substantially decrease the probabilities that microorganisms generate resistance32. Additional studies are necessary to confirm such a proposal.The 50 values for derivatives are still high, such information will be used to improve the reactivity of molecules synthesized in new refinement cycles and performing more medicinal chemistry studies. The ability of compounds to have multi-target activity in G. lamblia must be explored in order to identify their novel targets; such a characteristic appoint to these compounds as more-efficient anti-parasitic drugs.In conclusion, we demonstrate that gastric proton-pump inhibitors can be used as scaffold in drug-development to design better anti-parasitic molecules. Our data also offer a molecular mechanism of reaction in which the kind of adducts generated, together with the covalent bond that link to them with Cys residues have been clearly explained. Since the ICStarting materials, Glyceraldehyde 3-phosphate (GAP), solvents, starting materials and other analytical grade reagents were purchased from Sigma-Aldrich, Merck, JT Baker and Amresco. The α-glycerol 3-Pi dehydrogenase and NADH were purchased from ROCHE. Calf serum and molecular biology reagents were supplied from Thermo Scientific, New England, Invitrogen and Applied Biosystems. Growth medium of trophozoites was purchased from DB and Sigma-Aldrich.1H 13C and 2D NMR spectra were recorded in Bruker Avance 400 spectrometer  and Bruker 700 Acend III spectrometer . The chemical shifts are reported in ppm relative to the solvent resonance as the internal standard . Mass spectrometry analysis was assayed on a JEOL JMS-700 spectrometer as previously showed20.Melting points were determined in an EZ-Melt MPA 120 automated melting point apparatus and the results are uncorrected. Reactions were monitored by thin layer chromatography using 0.2 mm precoated silica gel plates . The H-benzimidazole core. A methyl group at position 5 of benzimidazole ring replaced the methoxy group, the rest of substitutions were carried out on the pyridine ring with a common pattern: an alkoxy substituent at position 4 of pyridine ring, and a methyl group at position 3. Compounds were designed and synthesized in the Laboratorio de Química Farmacéutica, Universidad Autónoma del Estado de Morelos. We firstly prepared the precursors 5-methyl-2-[(pyridin-2-ylmethyl)sulfanyl]-1H-benzimidazole through of nucleophilic substitution (SN2) among the 5-methyl-1H-benzimidazole-2-thiol and a properly substituted 2-chloromethyl pyridine hydrochloride under a strong alkaline media. We monitored the total conversion of starting materials to pyridinsulfanylbenzimidazoles by TLC assays. After that, precursors were smooth oxidized with 3-chloroperoxybenzoic acid at 0 °C; this reaction was monitored every 30 minutes by TLC in order to prevent the superoxidation, until obtaining the total conversion of precursors to the corresponding sulfoxide product. Afterward, all analogues were purified by silica gel column chromatography; the analogues labeled as BHO1, BHO2 and BHO3 were stored at 4 °C until their use. Stock solutions of analogues were prepared by dissolving to them in DMSO or ethanol. Stock concentrations of BHO1, BHO2 or BHO3 were calculated from their absorbances at 299 nm under alkali conditions, using molar absorption coefficient (ε M1−cm−1) values of 15476, 17304 and 13790, respectively. Omeprazole concentration was determined at 306 nm with ε M−1cm−1 of 15474.Three new compounds were designed on the basis of the structure of the proton-pump inhibitors omeprazole and lansoprazole, maintaining the 2-(2-pyridylmethylsulfinyl)-11H NMR  δ: 2.43 , 4.69–4.76 , 7.11 , 7.30 , 7.31 , 7.41 , 7.53 , 7.75 , 8.51  13.31  ppm. 13C NMR  δ: 21.3 (CH3); 61.9 (CH2); 123.2 (C-5′); 124.8 , 125.3 (C-3′); 132.8 ; 136.9 (C-4′); 149.7 (C-6′); 150.7 (C-2′); 153.2 (C-2) ppm. Carbons C3a, C7a, C4 and C7 were not observed due to tautomeric equilibrium. MS calculated for C14H13N3OS: 271.0779, found: 271.0779.Light-brown solid. Yield 75%. Mp. 121.3–122.9 °C. 1H NMR  δ: 2.16 , 2.19 , 2.43 , 3.68 , 4.68, 4.75 , 7.12 , 7.42 , 7.53 , 8.17 , 13.4  ppm. 13C NMR  δ: 11.64 (CH3), 13.48 (CH3), 21.87 (CH3), 60.01(CH3O), 60.85 (CH2SO), 126.54 (C-5′), 127.15 (C-3′), 148.7 (C-2′), 149.83 (C-6′), 164.5 (C-4′); too broad resonances only detected through HMBC: 111.96 (C-4), 119.99 (C-7), 125.09 (C-6), 126.27, 133.1, 134.7, 142.18, 152.5 (C-2) ppm. MS calculated for C17H19N3O2S: 329.1198, found: 329.1197.Light-brown solid. Yield 70%. Mp. 221.4–222.8 °C. 1H NMR  δ: 2.16 ; 2.44 ; 4.73, 4.81 , 4.90 , 7.09 , 7.12 , 7.48 , 7.57 , 8.28 , 13.42  ppm. 13C NMR  δ: 11.13 (CH3), 21.86 (CH3), 60.92 (CH2SO), 65.71 (CH2O), 106.15 (C-5′), 123.54 (C-3′), 148.53 (C-6′), 150.69 (C-2′), 161.99 (C-4′); too broad resonance only detected through HMBC: 111.7 (C-4), 120.11 (C-7), 124.8 (C-6), 126.9, 134.45 ppm. MS calculated for C17H16F3N3O2S: 383.0915, found: 383.0915.Light-brown solid. Yield 75%. Mp. 228.5–229.8 °C. 24. The recombinant GlTIM containing a His-tag in its NH-terminal together with a recognition region for tobacco each virus protease, was expressed in Escherichia coli. The GlTIM purification was performed by using IMAC method; after His-tag was removed33, the enzyme was stored at 4 °C in a buffer containing 100 mM triethanolamine, 10 mM EDTA, pH 7.4 (TE buffer). GlTIM purity was higher than 95% as determined by SDS-PAGE and MALDI-ToF mass spectrometry. Mutant of GlTIM C222A and human TIM (HsTIM) were prepared as described before33. Protein content of GlTIM and HsTIM was calculated by using a ε M−1cm−1 of 26,600 and 32,595, respectively. For all assays, the enzymes were freshly prepared with no more of two weeks of storage.For all assays we used mutant GlTIM C202A in order to work with the dimeric form of enzyme; this mutant was referred in the paper as GlTIM and was prepared as described before34. The reaction mixture contained TE buffer (pH 7.4) supplemented with 0.2 mM NADH, 1 mM GAP, and 0.9 U of alpha-GDH. The reaction started by adding 5 ng/mL of TIM.TIM activity was determined in a Cary50 spectrophotometer (Varian) at 25 °C using coupling assay following the conversion of D-glyceraldehyde 3-phosphate to dihydroxyacetone phosphate as described beforek1) values at each fixed concentration of compounds by fitting them to an exponential decay. Then, the k1 values were re-plotted against the employed concentrations of each compound; the second-order rate constant value of inactivation was obtained from the slope of these plots35.Omeprazole and compounds stock solutions were freshly prepared using 100% DMSO as solvent. For assays, TIM’s  were incubated in TE buffer (pH 7.4) at 37 °C with several concentrations of compounds; DMSO was maintained at 5% during incubation, this concentration did not affect the enzyme activity (data not shown). The reaction was stopped by diluting the enzyme 40,000 times in TE buffer and then, the remaining activity was determined. The second-order rate constants of inactivation were calculated from obtaining the pseudo-first order rate constant . Cysteine quantification was performed spectrophotometrically using de Ellman’s reagent as described before21. Thermostability of modified GlTIM was evaluated recording the loss of CD signal at 222 nm during a temperature scan ranging from 25 to 90 °C and an increase of 1 °C/2.5 min. The unfolded fraction of enzyme and melting temperature (Tm) was determined as reported before21.Changes in secondary structure of proteins were monitored by circular dichroism (CD) in a Jasco-810 spectropolarimeter containing a thermostated Peltier-controlled cell holder. For CD experiments, the enzymes were previously equilibrated in 25 mM phosphate buffer, pH 7.4. Spectral scans from 190 to 280 nm at 0.1 nm intervals were performed at 25 °C using a quartz cell with 0.1 cm of path length. All CD data were reported as molar ellipticity using the expression described beforeChanges of tertiary structure were analyzed following the protein intrinsic fluorescence in a Perkin-Elmer LS-55 spectrofluorometer. After proteins were excited at 280 nm, we recorded the emission spectra from 310 to 500 nm with a scan speed of 150 nm/min and using excitation and emission slits of 5 and 4 nm, respectively. We used phosphate buffer without protein as blank spectra and was subtracted from all fluorescence data. Changes in quaternary structure were evaluated by size exclusion chromatography (SEC) employing a Perkin-Elmer series 200 HPLC and a silica gel base column (YMC-Pack Diol-120) previously equilibrated with a mobile phase containing 150 mM NaCl, 50 mM potassium phosphate, pH 7.0. The system was calibrated using a gel filtration standard kit (Bio-Rad). Flow was maintained at 0.1 mL/min, the elution of proteins was monitored by their absorbance at 280 nm.BHO2 and BHO3) during 2 hours in TE buffer at 37 °C. Then the samples were incubated with 2 mM of DTT during 10 min at 25 °C; the samples were later centrifuged at 15,000 × g/15 min at 25 °C and supernatant filtrated by 3 mL Sephadex G-25 column equilibrated with Milli-Q water. After that, the samples were totally desiccated and sending to the CBC-UIC Research Resource Center Mass spectrometry, Metabolomics and Proteomic Facility  for performing an LC-MS/MS analysis. The proteins were digested by trypsin and the resulting peptides were applied to a Dionex Ultimate 3000 HPLC system coupled to a Thermo Fisher Scientific Orbitrap mass spectrometer. The obtained data were analyzed by using MAXQUAT Software.GlTIM  was incubated with 1 mM of compounds . We added five new post-translational modifications belonging to the compounds  and “B”  were added. For BHO2 and BHO3, their corresponding adducts “A”  were added. Finally, only one adduct “B” was added since BHO2 and BHO3 generate the same one . In addition, the variable modifications of methionine oxidation (Δm 15.9949), and asparagine deamidation (Δm 0.9840) were also included. The m/z tolerance values of the precursors and fragments were 10 ppm and 0.9 Da, respectively. From suite, X!Tandem version 2015.12.1538, MyriMatch39, MS-GF + 40, OMSSA41 and Comet42 were used as search engines. Modifications identified were confirmed by manual analysis by using MS-Product bundled in Protein Prospector Suite .We performed an additional in-house strategy of analysis by using the Peptide Shaker suite V 1.12.2).12.237. G. lamblia WB reference isolate in all experiments. The splitting method used to our susceptibility assays is described below. Cultures with 100,000 trophozoites/mL on TYI-S-33 medium were incubated during 48 hours at 37 °C supplemented with increasing concentrations of compounds ranging from 32.5 to 500 µM. Afterward, samples were chilled in ice for 15 minutes, washed with medium and resuspended in 50 µL of medium free of compounds and antibiotic. Then, trophozoites were diluted in 1 mL of medium free of compounds and antibiotic, incubated for 48 hours at 37 °C and then, the viability of trophozoites was manually quantified by using a Neubauer chamber; the viability percent and IC50 was subsequently calculated.We used the 2. After the cell cultures grew to confluence, we determined the cell viability by using Trypan Blue method; then, cells were seeded at 4 × 104 cells/well in 100 µL culture medium during 24 hours. Afterwards, the compounds were added in a volume of 100 µL using serial dilutions ranging from 500 µM to 31.2 µM; then, the cultures were incubated during 24 hours. DMSO was maintained constant at 0.2%; a control was performed by incubating cells with this solvent concentration but in absence of compounds. After incubation time, we measured the cell viability by using MTT -2,5-diphenyl-2H-tetrazolium bromide; Roche Diagnostics) method. Briefly, 10 µL MTT (5 mg/mL) was added to cell cultures and incubated at 37 °C during four hours; then, 100 µL of solution containing 10% SDS and 0.01N HCl was added. Thereafter, the formazan products were spectrophotometrically quantified at 570 nm . The CC50 (median cytotoxicity concentration) and CI95  values from each experiment were determined from the concentration-percentage plots of cell viability obtained by logistic regression. Statistical analysis was performed using the SPPSS software (version 17.0).We used the cell lines VERO (ATCC:CCL-81) and gingival human fibroblasts . The assay procedure is described below. Cells were maintained in a DMEM medium supplemented with antibiotic-antimycotic (1X), L-glutamine (1X) and 10% fetal bovine serum. Cells were incubated in 25 mL plastic culture flasks (Nunc) at 37 °C and 5% COSupplementary material


In addition, the data match pseudo-second-order kinetic model and Langmuir model well. The computation results of adsorption thermodynamics and stoichiometric displacement theory of adsorption (SDT-A) prove that the adsorption process is spontaneous and endothermic. Finally, IB possesses a higher selectivity adsorption to Cd(II) and a better reuse capacity. The functionalized biochar could solidify designated ions stably.Biochar is an excellent absorbent for most heavy metal ions and organic pollutants with high specific surface area, strong aperture structure, high stability, higher cation exchange capacity and rich surface functional groups. To improve the selective adsorption capacity of biochar to designated heavy metal ions, biochar prepared by agricultural waste is modified via Ionic-Imprinted Technique. Fourier transform infrared (FT-IR) spectra analysis and X-ray photoelectron spectroscopy (XPS) analysis of imprinted biochar (IB) indicate that 3-Mercaptopropyltrimethoxysilane is grafted on biochar surface through Si–O–Si bonds. The results of adsorption experiments indicate that the suitable pH range is about 3.0–8.0, the dosage is 2.0 g·L Among all the heavy metal ions, cadmium is the most higher poisonous substance [It is well known that farmland, an important section of ecological environment, is closely related to the problems of resource, grain and environment ,2,3. Howral area . Meanwhiubstance , jeopardubstance ,9. Once ubstance . ConsequFor scavenging contaminants, the methods of biological treatment, adsorption, precipitation, membranes separation and ion exchange had been carried out ,12. AmonThe objective of this work is to explore the selective recognition performance of a biochar prepared by green waste using 3-Mercaptopropyltrimethoxysilane as the surface conditioning agent and Epoxy-chloropropane as the cross-linking agent via Ionic-imprinted Technique. To investigate the adsorption capacity and selective recognition performance of the selective recognition functionalized biochar (IB), the initial pH of Cd(II) solution, sorbent dosage, adsorption kinetics and adsorption thermodynamics are studied, and analyzed by X-ray diffraction (XRD) patterns, Fourier transform infrared (FT-IR), Zeta potential analysis and X-ray photoelectron spectroscopy (XPS).2·2/(5H2O)  were used in this study.Biochar  was used in this study as the substrate material to prepare the ion-imprinted functionalized sorbent. All chemicals were analytical grade, and the water used in all experiments was deionized water (18.25 MΩ·cm). 3-Mercaptopropyltrimethoxysilane , Epoxy-chloropropane , and CdCl−1 hydrochloric acid were stirred for 8 h, then the solid product was recovered via filtration, and washed by deionized water to pH = 6.0, and dried under vacuum at 70 °C for 8 h. The sample was denoted as activated biochar (AB).Exactly 4.0 g biochar (100 mesh) mixed with 200 mL 6.0 mol·L2·2/(5H2O) were dissolved in 80 mL alcohol (95%) with stirring and heating at 50 °C, and then 4 mL MPS was injected in and reacted for 2 h. Next, 10.0 g activated biochar was added in the mixed solution, and the mixture was reacted for 20 h at 80 °C under stirring, recovered by filtration and washed 3 times with ethanol. Then, the sample was stirred for 2 h in 100 mL 6 mol·L−1 hydrochloric acid, and recovered via filtration, washed by 0.10 mol·L−1 NaHCO3 and deionized water up to the eluent pH = 6.0–7.0, dried under vacuum at 80 °C for 12 h, and denoted as imprinted biochar (IB). For comparison, the non-imprinted functionalized biochar was also prepared using an identical procedure, but without the addition of CdCl2·2/(5H2O), and denoted as non-imprinted biochar (NIB). The preparation procedure of IB is shown in Exactly 1.92 g CdCl−1 Cd(II) solution in 100 mL conical flask. The pH of Cd(II) solution was adjusted to the range of about 1.0–14.0 by 0.1 mol·L−1 HCl or 0.1 mol·L−1 NaOH solutions, and then the mixtures were agitated at 298.15 K for 1440 min. Afterwards, to investigate the effect of sorbent dosage, the IB dosage was changed from 0.5 to 5.0 g·L−1 with 50.00 mL 0.10 mmol·L−1 Cd(II) solution in 100 mL conical flask which pH about 5.0–6.0. The adsorption kinetics was determined by analyzing adsorption capacity at different time intervals (5–1440 min) with the same sorbent dosage (2.0 g·L−1) and initial Cd(II) concentration  in 100 mL conical flask with pH about 5.0–6.0. For adsorption isotherms, different concentrations of Cd(II) solution  were agitated until equilibrium was achieved with the same sorbent dosage (2.0 g·L−1) in 100 mL conical flask with pH about 5.0–6.0. The temperature factor was investigated by determining the adsorption capacity at 298.15 K, 303.15 K, and 308.15 K. In all adsorption experiments, the mixtures were separated by 0.45 μm filter membrane and the Cd(II) concentrations were measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES) .The methodology for adsorption experiments is as follows. All adsorption experiments were executed using an air thermostatic shaker , including the factors of initial pH of Cd(II) solution, sorbent dosage, contact time, initial Cd(II) solution concentration and adsorption temperature. The effect of initial pH of Cd(II) solution was carried out by dispersion of 0.1000 ± 0.0002 g IB and 50.00 mL 0.10 mmol·L−1 Cd(II)/Cu(II), Cd(II)/Zn(II), Cd(II)/Co(II) and Cd(II)/Pb(II) at pH 5.0–6.0 in 100 mL conical flask. After adsorption equilibrium, the concentration of each ion in the remaining solution was measured by ICP-AES. Cd(II) was desorbed by the treatment of with hydrochloric acid. In this section, 0.1000 ± 0.0002 g employed IB with 50.00 mL 6.0 mol·L−1 hydrochloric acid in 100 mL conical flask was agitated for different durations  by a magnetic stirrer. The final Cd(II) concentration in the aqueous phase was measured by ICP-AES. The ratio of desorption was calculated from the amount of Cd(II) adsorbed on IB and the final Cd(II) concentration in the desorption medium. Finally, all experimental results are the averages of thrice repeated experiments.The selective recognition adsorption experiments of Cu(II), Co(II), Pb(II) and Zn(II) ions with respect to Cd(II) were conducted using IB Biochar-based (BS) and NIB. Next, 0.1000 ± 0.0002 g sorbent were added in 50.00 mL metal ions mixed solution containing 0.10 mmol·L−1 by Spectrum One (Version BM) FT-IR  spectrometer with 32 scans resolution of 2 cm−1. Approximately 10% (mass fraction) of the solid sample was mixed with spectroscopic grade KBr. The Zeta-potentials of the fresh biochar and ageing biochar were measured using a Zetasizer Nano Zs90  at room temperature (25 °C) [XRD measurements were performed using an X-ray Diffractometer  with a Cu-Kα (λ = 0.15418 nm) radiation source. A continuous scan mode was used to collect the 2θ scan XRD data from 5° to 70° at the scanning speed of 5°/min; the voltage and current of the source were 40 kV and 40 mA, respectively. The XPS analyses were performed using a Kratos AXIS Ultra XPS system  equipped with a monochromatic Al X-ray source at 150 W. Each analysis started with a survey scan from 0 to 1350 eV with a dwell time of 8 s, pass energy of 150 eV at steps of 1 eV with 1 sweep. For the high-resolution analysis, the number of sweeps was increased, the pass energy was lowered to 30 eV at steps of 50 meV, and the dwell time was changed to 0.5 s. FT-IR spectra were collected in the range of 4000–400 cm (25 °C) . They weTo investigate the mechanism of Cd(II) adsorption on IB, two kinetic models ,20,27, pk1 is constant rate (min−1), and eq and tq are Cd(II) adsorption amounts (mg·g−1) at equilibrium t and time t (min), respectively.The pseudo-first-order kinetic equation:k2 is constant rate (min−1), and eq and tq are the Cd(II) adsorption amount (mg·g−1) at equilibrium t and time t (min), respectively.The pseudo-second-order kinetic equation: Langmuir and Freundlich models were used to describe the adsorption process ,20,27.eC is the equilibrium concentration (mg·L−1), eq is the equilibrium capacity of Cd(II) on the IB, mq is the monolayer adsorption capacity of the sorbent (mg·L−1), and b is the Langmuir adsorption constant (L·mg−1).Langmuir adsorption isotherms equation: fK and n are the Freundlich adsorption constant which indicate the adsorption capacity and intensity, respectively; and eq is the equilibrium capacity of IB to Cd(II).Freundlich adsorption isotherms equation: G0), Enthalpy change (ΔH0), and Entropy change (ΔS0), calculated using the following equations:dK is the distribution coefficient, T is the temperature (K), and R is the gas constant (8.3145 J·mol−1·K−1).The data of adsorption isotherms were used to estimate the thermodynamic parameters, Gibbs free energy change (ΔdK (dK = eq/eC) is the partition coefficient of solvent in liquid solid phase, eC is equilibrium adsorption concentration (mg·L−1), β is a constant that measures the affinity of the solute to the sorbent, Z represents the total moles of the solvent released or adsorbed for 1 mol solute together with its corresponding contact area on the adsorbent surface during the adsorption or desorption process, is q is the reduced molecule number of the solvent.The stoichiometric displacement theory of adsorption equation is as follows.β and q/Z are constant, while β and q/Z are linear to 1/T.When the temperature is invariant, β isThe definition of q/Z isβ and q/Z are obtained by the slope and intercept of the straight line plotting lgdK versus lgeC.The definition of TG, ΔTH, and ΔTS are calculated as follows.Δpdf = No. 41-1476), quartz (pdf = No.46-1045), graphite (pdf = No. 41-1487), calcite (pdf = No. 05-0586) and brushite (pdf = No. 09-0077), respectively, and also that the main mineral in AB was quartz (pdf = No. 46-1045). The comparison indicates that interfering metallic materials on the carbon surface are removed and the silicon content is relatively enhanced.−1 and 2853 cm−1 reflect –CH2 stretching vibration. A broad peak is noted at 1095 cm−1, due to the Si–O–Si, which shifted from 1048 cm−1 and indicates MPS interacted on the Si–O site on the surface of activated biochar. The absorption band at 2152 cm−1 is assigned to S–H vibrations of sulfydryl group.The FT-IR spectra of MPS, AB and IB are shown in q960 was similar to q1440). In R2 = 0.9964), and the parameters of pseudo-second-order kinetics are fitted. The calculated eq (6.76 mg·g−1)from the pseudo-first-order kinetic model agrees very well with the experimental data. Thus, the adsorption process is a chemisorption process.The equilibrium adsorption isotherm is fundamental in describing the interactive behavior between solute and sorbent, and it is important for the design of adsorption system. R2) obtained by linear regression. It indicates that the adsorption process is well described by the Langmuir model  adsorption on IB.rsus 1/T d. In TabH, ΔS and ΔG cannot be calculated accurately. Recently, stoichiometric displacement theory of adsorption (SDT-A) [However, these gas–solid adsorption equations were originally derived only for gas–solid adsorption systems, and they have not been related to the strong interactions that exist among the solute, solvent and solid sorbent in a liquid–solid adsorption process. The adsorption mechanisms of liquid–solid systems are more complex than those in gas–solid systems. In addition, the volume of the adsorption layer on solid sorbent surface cannot be estimated accurately, so the values of Δ (SDT-A) ,30,31 haAG indicates that Cd(II) adsorption on the IB was a spontaneous and heat release process, while increasing temperature benefits the adsorption. ΔTG was negative and decreases, indicating that the increasing temperature was beneficial to the Cd(II) adsorption. (2) At the same adsorption temperature, the constant ΔAG and ΔAH indicate that the adsorption process was a spontaneous and exothermic process, which was unaffected by the Cd(II) concentrations. (3) Under different conditions, the function variable of ΔTS indicated that the degree of disorder of the whole system increased. Compared to the thermodynamic parameters derived from Langmuir, the values of ΔG varied little, however, the values of ΔH and ΔS greatly differ.The data in −1), and there is a sharp decrease at pH > 8.0. At different pH environments, Cd(II) solution exhibited in different forms during the adsorption process, and the different forms affected the adsorption behaviors. The Cd(II) solution chemistry could be calculated to generate the concentration logarithmic diagram of each component at different pH. The relative species distribution of cadmium is calculated from the hydrolysis constants  [2(s) (spK = 5.27 × 10−15) and the initial Cd(II) concentration (1.0 × 10−4 mol·L−1). Cd(II) is found to form precipitation at pH ≈ 7.75 (the equations are shown in the C-pH of the Cd(II) hydrolysis components as a function of pH when the Cd(II) concentration is 0.1 mmol·L−1. It indicates that the main component at pH = 3.0–8.0 is in the form of Cd(II) ion, while the main component is Cd(OH)2(s) at pH > 8.0, when the reaction with IB is week. At lower pH (<3.0), the Cd(II) has to compete with hydrogen ion among the exchange sites. = 8.62) . The pre−1  is shown in −1 . With hiη = IBk:NIBk) for Cd(II)/Cu(II), Cd(II)/Zn(II), Cd(II)/Co(II) and Cd(II)/Pb(II) are 6.06, 5.98, 6.81 and 8.05, respectively, and the relative selectivity coefficients (η = IBk:kBS) are 5.37, 5.85, 6.50 and 6.10 (Competitive adsorption of Cd(II)/Cu(II), Cd(II)/Zn(II), Cd(II)/Co(II) and Cd(II)/Pb(II) were investigated in their double mixture systems. Relative selectivity coefficients (and 6.10 .The results indicate that IB has a higher selectivity for Cd(II), even in the presence of Co(II), Pb(II), Zn(II) and Cu(II) interferences in the same medium due to the coordination geometry selectivity of IB, which could provide ligand groups arranged in a suitable way for coordination of Cd(II). Although some ions have similar size with Cd(II) and some ions have high affinity with the ligand, IB still exhibits high selectivity for extraction of Cd(II) in the presence of other metal ions. The results also indicate that the adsorption capacity and selectivity of biochar based (BS) is low.Desorption of Cd(II) from IB is studied in a batch experimental set-up. The best desorption time is found to be 8 h. With a single washing, desorption ratio is up to 89.19% .To verify the reusability of IB, the cycle of adsorption–desorption was repeated seven times using the same sample. The results reveal that IB could be used repeatedly without significantly losing its adsorption capacities. Adsorption capacity of IB decreased only 15.64% after five adsorption–desorption cycles .−1. During the initial 120 min, the removal of Cd(II) from IB was rapid, and the equilibrium time was about 960 min. The adsorption process matched well with pseudo-second-order kinetic model and Langmuir model. The results of adsorption isotherms and SDT-A informed that IB adsorption capacity increased with the initial Cd(II) concentration and the adsorption temperature, and the adsorption process was spontaneous and endothermic. IB had a higher selectivity for Cd(II) in the presence of Co(II), Pb(II), Zn(II) and Cu(II), which provided ligand groups arranged in a suitable way for coordination of Cd(II). Overall, 6.0 mol·L−1 hydrochloric acid could remove Cd(II) from IB effectively, and the desorption ratio was up to 89.19% with a single washing. Adsorption capacity of IB decreased only 15.64% after five adsorption–desorption cycles.A biochar prepared by agricultural waste was modified using 3-Mercaptopropyltrimethoxysilane Epoxy-chloropropane via Ionic-imprinted Technique. IB adsorbed Cd(II) at suitable pH of about 3.0–8.0 and dosage of 2.0 g·L


Salty taste is one of the five basic tastes and is often elicited by NaCl. Because excess sodium intake is associated with many health problems, it could be useful to have salt taste enhancers that are not sodium based. In this study, the regulation of NaCl-induced responses was investigated in cultured human fungiform taste papillae (HBO) cells with five arginyl dipeptides: Ala-Arg (AR), Arg-Ala (RA), Arg-Pro (RP), Arg-Glu (RE), and Glu-Arg (ER); and two non-arginyl dipeptides: Asp-Asp (DD) and Glu-Asp (ED). AR, RA, and RP significantly increased the number of cell responses to NaCl, whereas no effect was observed with RE, ER, DD, or ED. We also found no effects with alanine, arginine, or a mixture of both amino acids. Pharmacological studies showed that AR significantly increased responses of amiloride-sensitive but not amiloride-insensitive cells. In studies using small interfering RNAs (siRNAs), responses to AR were significantly decreased in cells transfected with siRNAs against epithelial sodium channel ENaCα or ENaCδ compared to untransfected cells. AR dramatically increased NaCl-elicited responses in cells transfected with NHE1 siRNA but not in those transfected with ENaCα or ENaCδ siRNAs. Altogether, AR increased responses of amiloride-sensitive cells required ENaCα and ENaCδ. Taste papillae are divided into three morphological types, fungiform, circumvallate, and foliate papillae, which are located on the anterior, posterior, and lateral sides of the tongue, respectively3. Each taste bud contains at least four types of cells: types I–IV4. Type I cells express glutamate-aspartate transporters (GLAST) for glutamate. They also express NTPDase2, a plasma-membrane-bound nucleotidase involved in extracellular ATP hydrolysis, and ROMK, a potassium channel that may be responsible for maintaining K+ homeostasis. Type II cells express all the elements of the taste transduction cascade for sweet, bitter, and umami taste. Unlike type I and type II cells, type III cells express synaptic membrane proteins, neural cell adhesion molecule (NCAM), and synaptosomal-associated protein 25 (SNAP-25). Type IV cells are proliferative cells located at the bottom of the taste bud5. Taste plays a large role what we choose to eat, and there is a strong correlation between consumption of high-salt food and many health problems8. Currently, daily individual sodium consumption in most countries is reported to be more than twice the amount recommended by the World Health Organization9. Much effort has been made to decrease sodium consumption, but salt substitution has been limited mainly to infant formulas and baked foods11. As yet, no compounds are available that can effectively substitute for the taste of sodium chloride in food. Therefore, it is imperative to search for a salty taste enhancer as an alternative approach to reduce sodium consumption in the general population.Humans perceive five basic tastes – bitter, sweet, umami, sour, and salty – via taste receptor cells clustering in the taste buds of specialized papillae in the oral cavity13. Amiloride and its derivative benzamide are high-affinity blockers of ENaC7. In rodents, approximately 65% of fungiform papillae taste cells exhibit functional amiloride-sensitive Na+ currents, whereas only 35% of foliate papillae cells are amiloride-sensitive. In contrast, taste cells of the circumvallate papillae are completely insensitive to amiloride, although ENaCα mRNA and immunoreactivity to the purified amiloride-sensitive Na+ channel proteins have been detected in those cells3. The amiloride-sensitive pathway is Na+ specific and mediated by taste receptor cells expressing ENaC, a member of the degenerin/epithelial sodium channel (DEG/ENaC) family of non-voltage-gated ion channels15. However, the amiloride-insensitive pathway is cation nonselective, recognizing Na+, K+, and NH4+ salts16.Salty taste is recognized by salt receptors in the oral cavity, and evidence indicates that epithelium sodium channel (ENaC) subunits may play roles in this recognition and that at least two pathways, amiloride-sensitive and amiloride-insensitive, are involved in salty taste transduction18. Much less is known about the tastes of dipeptides, made of two amino acids joined by a planar peptide linkage, and there is no strict relationship between the taste of dipeptides and the constituent amino acids20. Previous reports indicate that arginine amino acid and the arginyl dipeptides Ala-Arg (AR), Arg-Ala (RA), and Arg-Pro (RP) may enhance salty taste, increasing the salty taste of 50 mM NaCl in both aqueous and model broth solutions in human sensory evaluations9. However, the underlying cellular mechanism is not known.Amino acids interact with many receptors; the tastes of individual amino acids are complex and in human sensory studies are described by more than one taste characteristicIn this study, we used cultured human taste cells to explore the mechanisms underlying the previously reported enhancement of salty taste by alanyl-arginine peptides. We examined effects on cellular responses to NaCl elicited by five arginyl dipeptides: AR, RA, RP, Arg-Glu (RE), and Glu-Arg (ER); and two non-arginyl dipeptides: Asp-Asp (DD) and Glu-Asp (ED). We found that the AR arginyl dipeptide increased the number of NaCl-induced responses, acting on amiloride-sensitive cells, targeting ENaCα and ENaCδ receptors. This work provides mechanistic information on the enhancement of NaCl-elicited responses by dipeptides and thus may suggest some alternatives to reduce sodium consumption.2+ responses elicited by dipeptides in cultured human fungiform taste (HBO) cells. Dipeptides at different concentrations  elicited a concentration-dependent response relationship, with EC50 values of 45, 122, 106, 89, 114, 100, and 82 µM for AR, RA, RP, RE, ER, DD, and ED, respectively  in the mixture. However, no effect on response frequency was observed at 50 mM NaCl. Neither 50 nor 150 mM NaCl-elicited responses were increased by 100 μM RP Fig. . Also, bFinally, alanine (Ala) and arginine (Arg), the component amino acids of the most active dipeptide AR, were further investigated alone and as a mixture for potential effects. Neither alanine nor arginine alone at concentrations of 10, 50, and 100 μM increased cell responses when mixed with NaCl , sweet mixture, and bitter mixture. The stimuli were applied sequentially but in random order. Responses to basic taste stimuli and to AR are shown in Fig. The breadth of tuning of cells responding to AR and NaCl was also investigated. At least one stimulus elicited a response in 102 of 679  taste cells. Of these 102 responding cells, 30 (29.4%), 62 (60.8%), and 10 (9.8%), respectively, responded to 50 μM AR alone, 150 mM NaCl alone, and both AR and NaCl Fig. . Of the In summary, the sensory response profile of AR-responsive cells was distinct from those that responded to umami, sweet, bitter, and salty stimuli.To investigate which receptor interacts with AR, we recorded HBO cell responses to AR with the specific blockers amiloride (ENaC blocker), cariporide , Ki16425 (lysophosphatidic acid receptor 1 [LPAR1] blocker), NPS2143 , and U73122 (phospholipase C beta 2 [PLCβ2] blocker).As a control, amiloride significantly inhibited the number of HBO cells that responded to 150 mM NaCl. The number of NaCl-responding cells were reduced by 70.5%, from 11.2%  to 3.3%  and 2.4%  of total taste cells, respectively. There was no significant difference in numbers of responding cells between the mixture of NaCl, AR, and amiloride  and the the calculated sum of responses to the individual compounds  of cells responding to AR alone or the mixture of AR and cariporide Fig. . Ki16425Amiloride significantly inhibited the number of cells that responded to either AR or the mixture of NaCl and AR. To confirm that AR increased the number of NaCl-elicited responses via ENaC and to investigate which subunit(s) of ENaC were involved, ENaCα and ENaCδ were downregulated using small interfering RNAs (siRNAs). siRNA knockdown of NHE1 was used as a negative control.Quantitative real-time RT-PCR (qPCR) and immunocytochemistry were used to verify siRNA knockdown of gene and protein expression, respectively. qPCR indicated that ENaCα, ENaCδ, and NHE1 mRNA expression was reduced by siRNA by 80.1%, 48.7%, and 94.8%, respectively Fig. . ImmunocTo determine which ENaC subunit(s) might interact with AR, we tested the responses of siRNA-transfected cells. Treatment with siRNAs against ENaCα, ENaCδ, and NHE1 significantly decreased the numbers of taste cells responding to 150 mM NaCl alone from baseline of 11.7%  to 1.5% , 2.3% , and 1.3% , respectively Fig. . siRNAs Responses of taste cells to the mixture of NaCl and AR were reduced from 24.8%  to 5.1% , 3.8% , and 8.0% , by siRNAs to ENaCα, ENaCδ, or NHE1 was respectively Fig. . No signENaCα siRNA did not alter the number of cells responding to the NaCl and AR mixture  vs. the sum of the individual responses  Fig. . SimilarThe pharmacological results indicated that another pathway might be involved in AR enhancement of cellular responses to NaCl. We next confirmed this hypothesis using ENaCα, ENaCδ, and NHE1 siRNA-transfected cells. No significant differences were observed between NaCl and the mixture of NaCl and amiloride, between AR and the mixture of AR and amiloride, or between the mixture of NaCl and AR and the mixture of NaCl, AR, and amiloride, in both ENaCα and ENaCδ siRNA-transfected cells Fig. . These r22, few studies have focused on human sensory evaluation of the taste of dipeptides19. In our study, we observed that dipeptides elicited intracellular calcium changes in HBO cells at the micromolar level , primarily elicits sweet taste but both sweet and umami tastes at higher concentration (600 mM)17. Similarly, L-arginine at 5 mM mainly tastes bitter, but sweet taste can be detected at 10 mM17. The tastes of dipeptides are more complicated compared with single amino acids. There is no strict relationship in terms of taste between dipeptides and their constituent amino acids. At millimolar concentrations, arginyl dipeptides AR, RA, and RP elicit bitter taste, whereas RE and ER exhibit no taste at all; the non-arginyl dipeptides DD and ED elicit sour taste20.Although amino acids elicit taste on cellular, molecular, and behavioral levelsvel Fig. , suggest9. Our results show that arginyl dipeptides, particularly AR and RA (50 µM), increased the number of HBO cells responding to NaCl  and use of different cell lines (αβγ-ENaC- or δβγ-ENaC-expressing oocytes vs. HBO cells) might explain the discrepancy.No study has evaluated the enhancement of NaCl-induced responses by arginyl dipeptides. Using patented HBO cultured human fungiform taste papillae cells, we have demonstrated that three out of five arginyl dipeptides  increased the number of cells responding to NaCl, but not the non-arginyl dipeptides tested  elicited HBO cell responses that differed from those of sweet, bitter, umami, and salty stimuli Fig. , indicat+ transport of common pathway for sweet, bitter, and umami taste transduction27. LPAR1 initiates downstream signaling cascades through PLC, mitogen-activated protein kinase (MAPK), Akt, and Rho, which alter a range of cellular responses, including cell proliferation and survival, cell-cell contact through serum response element activation, cell migration and cytoskeletal changes, Ca2+ mobilization, and adenylyl cyclase inhibition28. CaSR is a class C G-protein-coupled receptor that is moderately activated by the aromatic amino acids  and weakly activated by Arg, Lys, Val, and Gly29. In addition, various extracellular CaSR agonists enhance sweet, salty, and umami tastes, whereas no taste exhibited by themselves31. No cell response suppression was observed when HBO cells were presented with taste protein inhibitors cariporide, Ki16425, NPS2143, and U73122  over other cations and particularly sensitive to low Na+ concentrations32. Four homologous ENaC subunits, named α, β, γ, and δ, have been identified in human. Among them, the subunits α (or δ), β and γ, which are highly conserved in all vertebrates, are essential for the assembly of functional channels. The δ ENaC subunit exhibits maximal activity when co-expressed with β and γ subunits34. Both pharmacological and siRNA studies indicated that AR increased NaCl-induced responses via the amiloride-sensitive pathways  has been proposed as the candidate amiloride-insensitive receptor, based on the observation that TRPV1-knockout mice had diminished taste nerve response of the amiloride-insensitive response to salty tasteaCl Fig. . HoweveraCl Fig. . Therefo2 2, MgCl2 1, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 20, and Na-pyruvate 1, was prepared along with Ringer solution, containing (in mM) NaCl 145, KCl 5, CaCl2 2, MgCl2 1, HEPES 10, and Na-pyruvate 1. Dipeptides AR, RA, RP, RE, ER, DD, and ED were synthesized using the solid-phase peptide synthesis technique and purified using reverse HPLC (NEOscientific) to achieve >98% purity and dissolved in Milli-Q water to prepare a stock of 100 mM. All amino acids used were L-isomers, and all dipeptides were composed of L-amino acids.NMDG Ringer solution, containing (in mM) NaCl 30, N-methyl-D-glucamine (NMDG) 115, KCl 5, CaCl40. Monopotassium glutamate  was used for umami taste, and NaCl (150 mM) was used as salt stimuli. In the pharmacological studies, the specific blockers amiloride (50 µM), cariporide (5 µM), Ki16425 (0.2 µM), NPS2143 (0.5 µM), and U73122  were used. All the solutions were freshly prepared and adjusted to pH 7.0 and 300–310 mOsm/Kg·H2O before use.A sweet mixture comprising 1 mM sucralose and 100 mM glucose was used to ensure responses were from both artificial and natural sweet tastants. A bitter mixture comprising 2 mM phenylthiocarbamide, 5 mM salicin, and 2 mM denatonium benzoate was used because these bitters interact with different bitter receptorsHealthy, 22- to 40-year-old men (n = 2) and women (n = 2) were screened as potential donors of lingual fungiform papillae. Procedures were approved by an institutional review board (IRB) at the University of Pennsylvania and carried out in accordance with relevant guidelines and regulations. Subjects provided written, informed consent on IRB approved forms prior to testing. Subjects excluded on initial screening included those reporting a medical history or current manifestation of systemic, chronic disease; those reporting the regular use of prescription medications; and those reporting dry mouth or oral disease and the oral surgeon’s opinion that the subject was not appropriate for the study. The oral cavity was inspected for general tissue health. Subjects meeting these criteria were invited to participate in the study.42. Briefly, human fungiform taste papillae were removed from the anterior of the tongue. The papillae were incubated in isolation buffer mixed with soy trypsin inhibitor, collagenase, and elastase and then transferred to Iscove’s modified Dulbecco’s medium (Gibco) containing 10% fetal bovine serum, a 1:5 ratio of MCDB 153  medium (Sigma), 1% antibiotic-antimycotic, and 0.015% gentamycin (2.5 µg/mL). Cells with medium were gently resuspended, transferred to a sterile tissue culture flask, and incubated at 36 °C in a humidified incubator containing 5% CO2. One-third of the culture medium was replaced after 48 h and again after 5–7 days.Taste cells were cultured as previously reported2+]i) in response to stimuli were measured using calcium imaging42. Cultured HBO cells were seeded onto coverslips, grown for 3–5 days, and then loaded with 5 µM fura-2 AM  and 10% Pluronic F127 (Life Technologies) dissolved in DMSO in NMDG Ringer solution for 1 h at 36 °C. The images were visualized with an inverted fluorescence microscope (Olympus) and a CCD camera (Photometrics). The stimulus delivery and removal were controlled by a two-channel peristaltic pump (Spetec). Cells were exposed to the stimulus for 1 min. The cells were provided with at least 5 min of recovery time between stimuli. Images were captured every 2 s during stimulus HBO applications, with excitation wavelengths of 340 and 380 nm and an emission wavelength centered at 510 nm. Cell focusing, defining regions, and image acquisition were controlled by Metafluor software (Molecular Devices).Changes in intracellular calcium levels  of each dipeptide were tested. Each peptide was applied in random order on HBO cells in independent experiments and responses to peptide were measured by intracellular Ca2+ changes. Data from each concentration of peptide were collected from independent experiments. To evaluate dipeptide modulating NaCl-induced responses, cells were consecutively stimulated with three different stimuli in the following order: dipeptide alone, NaCl alone, and a mixture of NaCl and dipeptide. The concentrations of AR and RA were 10, 50, and 100 µM; the concentrations of other dipeptides were 50 and 100 µM. Both 50 and 150 mM NaCl were examined. To investigate the breadth of tuning of AR, HBO cells were consecutively stimulated in order with AR, sweet mixture, bitter mixture, and MPG. In pharmacological studies, cells were stimulated with three stimuli in order: a mixture of NaCl and AR, a mixture of NaCl, AR, and amiloride, and then the mixture of NaCl and AR again, to demonstrate that cells were as responsive as before amiloride exposure. To investigate whether AR increased responses of amiloride-insensitive cells, HBO cells received three stimuli in order: a mixture of NaCl and amiloride, a mixture of AR and amiloride, and a mixture of NaCl, AR, and amiloride. In siRNA studies, both siRNA-transfected and untransfected cells were stimulated by NaCl alone, AR alone, and a mixture of NaCl and AR. To evaluate whether other pathways were involved in the AR effect, siRNA-transfected cells were stimulated by the following six stimuli: NaCl alone; a mixture of NaCl and amiloride; AR alone; a mixture of AR and amiloride; a mixture of NaCl and AR; and a mixture of NaCl, AR, and amiloride.For EC43. Briefly, HBO cells were seeded onto coverslips in 12-well plates. After reaching 70–80% confluence, cells were transfected with 30 nM of prepared siRNA premix diluted in taste cell medium. The medium was changed 24 h post-transfection. Transfection efficiency was determined using qPCR and immunocytochemistry. Transfected cells were tested after 72 h by calcium imaging.The ENaCα-, ENaCδ-, and NHE1-specific siRNAs (Qiagen FlexiTube Premix siRNA) were used to downregulate ENaCα, ENaCδ, and NHE1 expression, respectively. Scrambled siRNA (Qiagen) was used as a negative control. siRNAs were dissolved in RNase-free water following the manufacturer’s protocol. siRNA transfection was carried out as described previously40. HBO cells were transfected with siRNA and further incubated until 72 h post-transfection. Total RNA was extracted by a Purelink RNA Mini Kit (Life Technologies) following the manufacturer’s instructions. DNase treatment of RNA was conducted with the Turbo DNA-free kit (Life Technologies). RNA quality was measured using an Agilent RNA ScreenTape assay (Agilent Technologies). Total RNA was reversed transcribed into cDNA using high-capacity cDNA reverse transcription kits (Applied Biosystems). Relative quantitation of the expression of selected genes was performed using the QuantStudioTM12K real-time PCR system (Applied Biosystems) and ENaCα, ENaCδ, and NHE1 TaqMan gene expression assay kits (Applied Biosystems). PCR amplification was performed in a final volume of 20 µL containing 1 µL 20× TaqMan gene expression assay, 10 µL 2× TaqMan gene expression master mix, 4 µL cDNA, and 5 µL RNase-free water. The cycling conditions were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min. Results were normalized using GAPDH (Applied Biosystems).qPCR was conducted to determine knockdown efficiency42. HBO cells were transfected with ENaCα, ENaCδ, and NHE1 siRNA, and further incubated for 72 h. Untransfected and scrambled siRNA-transfected cells were used as controls. The cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then permeabilized with 10% methanol and 10% hydrogen peroxide in PBS for 15 min. Then cells were blocked with 3% goat serum, 3% bovine serum albumin in PBS for 1 h and stained with primary antibodies ENaCα and ENaCδ (LSBio) and NHE1 (Alomone Labs) overnight at 4 °C, followed by goat anti-rabbit secondary antibody  for 30 min at room temperature. Coverslips were mounted using Vectashield with 4’,6-diamidino-2-phenylindole  after washing with PBS and water. Substituting the primary antibody with host IgG was used to determine the absence of nonspecific labeling. The photo was taken using a Leica TCS SP2 spectral confocal microscope (Leica Microsystems).Immunocytochemistry confirmed that siRNA transfection successfully downregulated ENaCα, ENaCδ, and NHE1 expressionF340/F380) was recorded for regions of interest (ROIs) were selected for each cell. Increases in intracellular Ca2+ evoked by stimulus application were expressed by ΔF = FPeak − FBaseline, where F = absorbance at 340/380 nm. The criterion for considering a responding cell was ΔF ≥ 0.03. Cells that failed to return to baseline were not counted, nor were apparent responses that occurred within 30 s or more than 120 s after stimulation. The percentage of responding cells was calculated by dividing the number of cells with a detectable Ca2+ increase by the total number of cells in the given experimental condition. The baseline fluorescence (ΔF/F) value of a cell was measured before taste stimuli given and the peak value of Ca2+ was measured after taste stimuli given. Statistical analysis of Ca2+ changes was conducted by counting the Ca2+ peaks during the first 2 min of stimulation. For each of the experiments, “n=” indicates number of experiment performed. Data were processed and plotted using Origin 8 (OriginLab) and Excel (Microsoft). Statistical comparisons between experiments were performed using a one-way analysis of variance and Tukey’s post-hoc test using GraphPad Prism . p-Values ≤ 0.05 were considered significant.The change in fluorescence ratio (Supplementary information


ACTB, GAPDH, RNF7, RHOA, RPS18, RPL32, PPIA, H3F3, API5, B2M, AP1S1, DRAP1, TBP, WSB, and VAPB) in porcine skeletal muscle at 26 different developmental stages . We evaluated gene expression stability using the computer algorithms geNorm, NormFinder, and BestKeeper. Our results indicated that GAPDH and ACTB had the greatest variability among the candidate genes across prenatal and postnatal stages of skeletal muscle development. RPS18, API5, and VAPB had stable expression levels in prenatal stages, whereas API5, RPS18, RPL32, and H3F3 had stable expression levels in postnatal stages. API5 and H3F3 expression levels had the greatest stability in all tested prenatal and postnatal stages, and were the most appropriate reference genes for gene expression normalization in developing skeletal muscle. Our data provide valuable information for gene expression analysis during different stages of skeletal muscle development in mammals. This information can provide a valuable guide for the analysis of human diseases.The selection of suitable reference genes is crucial to accurately evaluate and normalize the relative expression level of target genes for gene function analysis. However, commonly used reference genes have variable expression levels in developing skeletal muscle. There are few reports that systematically evaluate the expression stability of reference genes across prenatal and postnatal developing skeletal muscle in mammals. Here, we used quantitative PCR to examine the expression levels of 15 candidate reference genes ( Gene expression analysis provides important information for the study of gene function. Reference genes are used to judge gene expression levels and changes in target gene expression. Quantitative PCR (qPCR) is an important method in evaluating gene expression, which was first invented by Applied Biosystems Corporation (USA) in 1996. This technology significantly advanced gene quantitative research, and provided high sensitivity, specificity, and accuracy . QuantitGAPDH, ACTB, and HPRT  muscles at 26 developmental stages  in Landrace pigs . The expression stability of these reference genes in the porcine muscle samples was evaluated using qPCR analysis and the expression analysis programs NormFinder  muscle samples were collected from Landrace fetuses on the following days post-coitum (dpc): 33, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, and 105 dpc. LD muscle samples were collected from piglets on the following days after birth (dab): 0, 10, 20, 30, 40, 60, 80, 100, 140, 160, and 180 dab. Three biological replicates were collected at each time point, and totally 78 samples were collected. All samples were immediately frozen in liquid nitrogen and stored at –80 °C until further processing. Total RNA was extracted using TRIzol Reagent  according to the manufacturer’s instructions. RNA quantity and quality was determined by the Evolution 60 UV-Visible Spectrophotometer (Thermo Scientific). RNA preparations with an A260/A280 ratio of 1.8–2.1 and an A260/A230 ratio > 2.0 were selected for this assay. RNA integrity was determined by analyzing the 28S/18S ribosomal RNA ratio on 1.5% agar gels. Only RNA preparations that resolved with three clear bands on these gels were used for the transcriptome sequencing and qPCR analysis.All animals were sacrificed at a commercial slaughterhouse according to protocols approved by the Institutional Animal Care and Use Committee at the Institute of Animal Science, Chinese Academy of Agricultural Sciences . For the purpose of identifying potential reference genes during skeletal muscle development, candidate reference genes were selected according to previous studies . The topThe cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions for reverse transcription (RT) PCR. A mixture of 2 µg of total RNA and 1 µL of random primer was incubated at 65 °C for 5 min to dissociate the RNA secondary structure. Next, the following reaction was carried out in a total volume of 20 µL: 12 µL of the first reaction mixture, 4 µL of 5× RT buffer, 2 µL of 10 nM dNTP, 1 µL of RevertAid Reverse Transcriptase (200 U/µl) inhibitor, and 1 µL RiboLockRNase Inhibitor (20 U/µl). The reverse transcription reaction was performed at 25 °C for 5 min, followed by 42 °C for 1 h and 5 min at 70 °C. The cDNA was then diluted 7-fold, and stored at −20 °C until use.B2M, RHOA, RPL32, DRAP1 RNF7, WSB, and H3F3  under the following cycling conditions: 95 °C for 5 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 45 s. Three independent individuals at each time point were used for temporal and spatial analyses. Each qPCR reaction was performed in triplicate for technical repeats. The mean quantification cycle (Cq) value was used for further analysis. The primer sequences were according to or based on those of previous reports as follows : B2M, RHand H3F3 ; ACTB, Aand VAPB ; GAPDH and RPS18 ; and TBPand PPIA .Gene expression data were transformed to relative quantities using geNorm and NormFinder. GeNorm provided a measure of gene expression stability (M) Mj = gene stability measure,Vjk = pairwise variation of gene j relative to gene k,n = total number of number of examined genes.Lower M values represent genes with more stable expression across specimens being compared and generated a ranking of the putative reference gene expression levels from the most stable  to the least stable . GeNorm also generated a pairwise stability measure, which can be used to evaluate the suitable number of reference genes for normalization.NormFinder provided a stability measure (SV), identified the most stable gene, and calculated the best combination of two reference genes. This program focuses on finding the two genes with the least intra- and inter-group expression variation or the most stable reference gene in intra-group expression variation. Genes with lower stability values show a stably expressed pattern, while the higher stability values share the least stably expressed pattern.The BestKeeper program was used to compute the geometric mean of each candidate gene’s Cq value, to determine the most stably expressed genes based on correlation coefficient (r) analysis for all pairs of candidate reference genes (≤10 genes), and to calculate the percentage coefficient of variation (CV) and standard deviation (SD) using each candidate gene’s crossing point (CP) value . In the BestKeeper program, genes with higher r values and lower CV and SD values are more stable reference genes.ACTB, API5, B2M, GAPDH, RNF7, H3F3, PPIA, AP1S1, PPIA, RHOA, RPS18, RPL32, TBP, WSB, and VAPB) in the LD muscle samples at 15 embryonic stages  and 11 postnatal stages  of Landrace pigs. To minimize experimental error, triplicate amplifications were performed for individual experiments. The Cq values were computed to quantify the candidate reference gene expression levels. A higher Cq value means lower gene expression levels. Analysis of gene expression stability was based on the Cq values generated by qPCR  and AP1S1 had the highest mean Cq value (26.36). All candidate reference genes were abundantly expressed in skeletal muscle and showed wide variations in expression levels at different developmental stages. Therefore, it was necessary to evaluate gene expression stability and determine the suitable number of reference genes for accurate gene expression profiling in developing skeletal muscle.We performed qPCR assays to measure the expression levels of 15 candidate reference genes  for the 15 candidate reference genes using the geNorm program. Genes with lower M values have more consistent expression levels. The M values of the candidate genes are presented in pression  in develvariable . When onvariable . The geNn/Vn+1 value ranged from 0.059 to 0.111, which were all lower than the cut-off value of 0.15  value between two sequential normalization factors; it has a cut-off value of 0.15 for the pairwise variation according to the previous study , below w of 0.15 . These r of 0.15 , and two of 0.15 . These rAPI5 was the most stable reference gene with the lowest stability value (SV = 0.088) in all tested developmental periods , CV, and SD values  and lower CV and SD values are considered as stable and suitable reference genes. In all tested developmental periods, API5 expression had the lowest CV value (2.09) and almost the lowest SD value (it was slightly larger than that of RPL32 expression). Therefore, we propose that API5 is the most suitable reference gene for expression analysis of developing skeletal muscle during the tested prenatal and postnatal stages. API5 also was selected as the most stable gene during the postnatal period, whereas VAPB was the most stable gene for the analysis of developing skeletal muscle during the embryonic period.Then, BestKeeper program was used to evaluate the reference gene expression stability. We used BestKeeper to identify the optimal reference genes on the basis of the correlation coefficient . Therefore, the results of our study are more robust and accurate.Many researchers have studied the suitable reference genes in pig skeletal muscle. For example, API5) was the best candidate reference gene for normalizing target gene expression in developing skeletal muscle across the tested prenatal and postnatal periods. API5 is highly conserved across species from microorganisms to plants and animals  showed that apoptosis inhibitor 5   , whereasRPL32, RPS18, and H3F3 were the most stable reference genes in 33, 65, and 90 dpc skeletal muscle in Landrace pigs (We previously reported that ace pigs . The curGAPDH and ACTB) were not suitable as reference genes for skeletal muscle development. Our results showed that API5, a newly discovered reference gene, was the most suitable reference gene for all tested periods and muscle samples. RPL32, RPS18, VAPB, and H3F3 also were suitable as reference genes in developing skeletal muscle. Our data provide a guide for choosing appropriate reference genes for studies on skeletal muscle development and diseases in humans and other mammals.Our study evaluated the expression stability of 15 candidate reference genes in LD skeletal muscle across 26 prenatal and postnatal developmental periods in Landrace pigs. We found that the commonly used reference genes (10.7717/peerj.2428/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.2428/supp-2Supplemental Information 2Click here for additional data file.