Results evidenced that 27/55 of them had significantly different values in IF with respect to those measured in CTRL. The metabolic pattern of these potential biomarkers of infertility was cumulated (in both CTRL and IF) into a Biomarker Score index , that fully discriminated CTRL  from IF . The Biomarker Score values were significantly higher than those of CTRL in each of the six subgroups of IF. Posterior probability curves and ROC curve indicated that values of the Biomarker Score clustered CTRL and IF into two distinct groups, based on the individual FF metabolic profile. Furthermore, Biomarker Score values correlated with outcome measures of ovarian stimulation, in vitro fertilization, number and quality of blastocysts, clinical pregnancy, and healthy offspring. These results strongly suggest that the biochemical quality of FF deeply influences not only the effectiveness of IVF procedures but also the following embryonic development up to healthy newborns. The targeted metabolomic analysis of FF (using empowered Redox Energy Test) and the subsequent calculation of the Biomarker Score evidenced a set of 27 low molecular weight infertility biomarkers potentially useful in the laboratory managing of female infertility and to predict the success of assisted reproduction techniques.Nearly 40–50% of infertility problems are estimated to be of female origin. Previous studies dedicated to the analysis of metabolites in follicular fluid (FF) produced contrasting results, although some valuable indexes capable to discriminate control groups (CTRL) from infertile females (IF) and correlate with outcome measures of assisted reproduction techniques were in some instances found. In this study, we analyzed in blind FF of 35 control subjects  and 145 IF (affected by: endometriosis,  Infertility is defined as the failure to achieve a clinical pregnancy after at least 12 months of regular unprotected sexual intercourse. Nearly 40–50% of infertility problems are estimated to be of female origin, and approximately 30% of cases are of male origin. A further 20–25% of couples suffer from unexplained infertility [When infertile females undergo in vitro fertilization (IVF), it is known that only 32% of IVF cycles result in pregnancy .FF is the biological fluid that supports the maturation of the oocyte, influences the effectiveness of oocyte fertilization, and affects embryo development of fertilized oocytes. FF is easily accessible for withdrawals during oocyte pick-up, is abundant, and, above all, it is currently considered a waste product. FF may represent an optimal biological sample to perform non-invasive biochemical analyses aimed to predict oocyte quality and, potentially, even that of an embryo.FF is a mixture of secretions from granulosa and theca cells and compounds diffusing across the basement membrane from plasma, including proteins, hormones, metabolites, and toxins . GranuloDue to the multiplicity of their biochemical functions, the determination of low molecular weight compounds in FF appears of particular relevance to highlight the quality of this biological fluid. This category of compounds includes substances directly (glucose) or indirectly (some amino acids) involved in catabolic reactions connected to energy production, catabolites of cell metabolism reflecting cell bioenergetic , water- and fat-soluble antioxidants involved in defense mechanisms towards oxidative/nitrosative stress, as well as waste products of potential toxicity to the oocyte .1H-NMR) [In recent years, several studies have been carried out to determine the metabolic FF profile of women with infertility due to different causes. Using an untargeted metabolomic approach, with the aid of high-resolution proton nuclear magnetic resonance (1H-NMR) ,10 or li1H-NMR) ,11, it h1H-NMR) , ROR [131H-NMR) ,15. Some1H-NMR) ,17,18. Aall-trans retinoic acid, all-trans retinol, α-tocopherol, γ-tocopherol, total carotenoids, malondialdehyde, 8-hydroxy-2′–deoxyguanosine, nitrites, nitrates, creatinine, cytosine, cytidine, uracil, β-pseudouridine, hypoxanthine, xanthine, uridine, inosine, guanine, and guanosine) are different from those recorded in control fertile males [In a multicenter study using a targeted metabolomic approach, we have previously shown that the concentrations of various low-molecular-weight compounds in seminal plasma of infertile males , of waste products (lactate and ammonia), and of catabolites related to energy metabolism . Additional aims of the study were to determine whether females with different clinical infertilities had different biochemical signatures of the aforementioned compounds in their FF and to assess whether biochemical differences in FF of infertile females correlated with the main outcome measures useful to assess successful IVF.The main clinical features of the FF donors, as well as the administration protocols for ovarian stimulation, are reported in p < 0.01). Significantly lower basal FSH serum levels were found in the subgroups of EM and PCOS. Pooled IF had significantly lower values of both the number of retrieved and mature oocytes. After categorization into the different clinical subgroups, PCOS only had values of both parameters equal to those measured in the group of CTRL.No age differences were observed by comparing the group of CTRL with that of pooled IF, whilst the subgroup of AR-ROR, as expected, was composed of patients significantly older than both CTRL and any other subgroup of IF .10 and total carotenoids), as well as of amino acids  having significantly different values in FF samples of the two groups of CTRL and IF (p < 0.001).Similarly, When the group of IF was categorized according to the differential diagnosis of infertility, allowing to obtain 6 subgroups of IF , it was possible to observe that the various subgroups had specific patterns of alterations in the values of the aforementioned compounds in their FF (p < 0.001). The box plot of the Biomarker Score values determined in the two groups is illustrated in Using the values of the 10–90% percentiles reported in As shown in It can easily be observed that any patient scoring a Biomarker Score value ≤ 9 belongs to the group of CTRL. Conversely, patients scoring a Biomarker value ≥ 11 can certainly be assigned to the group of IF. Further confirmation of the power of the Biomarker Score as a tool to discriminate the biochemical characteristics of FF is represented by the ROC curve shown in p < 0.001).The number of retrieved oocytes, mature oocytes, fertilized oocytes, blastocysts, high-quality blastocysts, and β-HCG positive in CTRL and IF patients are shown in the box plots of q < 0.001).By categorizing IF patients into the 6 groups of infertility (q < 0.05). The number of patients of all infertile subgroups, but PCOS and GI patients, resulting in positive to the β-HCG analysis were significantly lower than that of CTRL (q < 0.01). The Pearson’s correlation coefficients were calculated to evaluate whether the Biomarker Score influences the biological measures of IVF. To this purpose, all patients were combined into a single group of FF donors, since we assumed that the possible correlation between the Biomarker Score and the biological measures of IVF was independent of the patient’s clinical condition. As shown in p < 0.001), of mature oocytes , of fertilized oocytes , of blastocysts , and of high quality blastocysts , therefore, strongly indicating that biological parameters of IVF are inversely correlated with the Biomarker Score.PCOS patients differed from CTRL only for the number of high-quality blastocysts (p < 0.001 compared to CTRL) were β-HCG positive, 16/113 developed clinical pregnancies , and 11/109 generated healthy offspring . The influence of the Biomarker Score on outcome measures of effective IVF was assessed by considering patients with positive or negative β-HCG , those developing or not developing clinical pregnancies  and those who delivered or not delivered healthy offspring , regardless of the group they belong to (CTRL or IF). The Biomarker Score values of each patient were then paired to the corresponding outcome measures. Hence, comparisons were performed between the Biomarker Score values of patients with positive parameters (scoring 1) versus the Biomarker Score values of patients with negative parameters (scoring 0). The box plots in Differences in the outcome measures of clinical pregnancy in CTRL and IF were clearly evidenced by indices related to fertilization and procreation capacities. In the group of CTRL, 25/25 blastocysts were transferred (100%). Following implantation, 15/25 patients (60%) were β-HCG positive, 11/25 developed clinical pregnancy (44%), and 11/25 generated healthy offspring (44%). In the group of IF 113/114 blastocysts were implanted (99.1%). In this case, only 26/114 patients , including energy metabolism substrates and products, purines, pyrimidines, hydrophilic antioxidants, oxidative/nitrosative stress biomarkers, lipophilic vitamins and antioxidants, and amino acids, the present study demonstrated significant anomalies in the FF composition of IF  compared to those found in CTRL. In particular, 27/55 compounds in FF of IF had values significantly different from those measured in CTRL, and these differences were also observed when IF were categorized according to the clinical diagnosis of infertility.In the last decade, there was an increasing interest to evaluate the metabolic profile of FF in females affected by infertility in order to establish whether potential differences in the concentrations of specific compounds might be causative of, or related to infertility ,22. The p < 0.001) and 3.87 ± 0.99 mmol/L FF . With the exclusion of IF affected by PCOS and GI, the subgroups of those affected by EM, PCOS, AR-ROR, ROR, UI, and GI showed lower FF glucose values and higher FF lactate values than those measured in FF of CTRL (q < 0.005). There are no unanimous findings in previous studies on the concentrations of these energy-linked metabolites in FF of IF compared to those of CTRL. Results indicating either normal glucose and higher lactate [The first notable result of the present study concerned glucose and lactate, the concentrations of which in CTRL were, respectively, 3.00 ± 0.45 mmol/L FF and 2.55 ± 0.60 mmol/L FF. Differently, we found that these compounds in pooled IF were, respectively, 2.15 ± 0.62 mmol/L FF , encountered in all subgroups after the clinical IF categorization . Our results indicate that lower glucose availability in FF occurs in pooled IF and in the IF subgroups of EM, AR-ROR, ROR, and UI, whilst PCOS and GI have normal glucose FF levels. Differently, an increase in FF lactate concentrations occurred in all IF subgroups, therefore appearing as a common biochemical feature of IF. These findings suggest that an imbalance between glucose consumption and glucose oxidation , takes place during oocyte maturation in all IF, leading to an increase in the glycolytic rate with lactate overproduction. Some of the IF patients also showed a decrease in the efficiency of glucose transfer from the blood to FF. The energy deficit hypothesis seems to be corroborated by the 1.8 and 1.9 times increase in the concentrations, respectively, of hypoxanthine and xanthine in FF of IF , and a significant increase of MDA, 8-OH-dG, nitrite, and nitrate (p < 0.001). Interestingly, when categorizing the patients according to their clinical diagnosis, it was possible to observe common alterations of oxidative/nitrosative stress biomarkers but different patterns of antioxidant deficiencies, with EM and PCOS being more prone to show a decrease in fat-soluble antioxidants rather than in water-soluble antioxidants , when patients were categorized according to different clinical diagnoses of infertility, some of the six resulting subgroups included a small number of subjects. Therefore, it was not possible to discriminate the different subgroups of IF on the basis of their Biomarker Score values, even though it was possible to observe differences in specific FF metabolites characterizing some subgroups of IF . As an additional study limitation, it should be considered that effecting the follicle pick-up when the follicle size is smaller than ≥18 mm (the follicle size of the pick-ups of our FF donors) different FF metabolic profiles might possibly be found. A further limitation is that the present results were obtained by measuring the various metabolites in pooled FF for those donors in whom more than one follicle was retrieved. This methodological choice, while being probably of minor relevance in differentiating CTRL from IF and from each of the six infertile subgroups, certainly limited the possibility to grade follicles from the same donor on the basis of the single follicle FF biochemical profile. Lastly, it should also be taken into account that the FF metabolic profiles might have been influenced by the different ovarian stimulation protocols, thus potentially acting as a confounding factor in the result interpretation.Although the study produced very promising results, there are some limitations that should be clarified in the future. For instance, notwithstanding the number of pooled IF had a reasonable size  and IF into two distinct groups, but also correlated with biological outcome measures of oocyte and embryo development. Therefore, performing the metabolic profile of FF, and subsequently calculating the Biomarker Score value, would appear of great utility in the laboratory management of female infertility, also in light of potential personalized therapies aimed to selectively correcting the altered biochemical profiles of FF.The study was conducted according to the Declaration of Helsinki for Medical Research involving Human Subjects. Informed written consent was obtained from each FF donor enrolled in this study.n = 145) were unable to obtain pregnancy after at least one year of unprotected sexual intercourse. Control FF donors  were recruited among couples in which inability to obtain pregnancy was due to a male factor alone. Patients were included in the group of CTRL after a careful clinical assessment and only when the diagnosis of male infertility was clearly established. Patients were excluded from the study in the case of mechanical barrier in their reproductive system, previous history of cancer, and premature ovarian failure. Gynecological assessment, including hormone analysis, hysterosalpingography, and transvaginal ultrasound to evaluate the uterine cavity and antral follicle counts were used to categorize infertile females into the following groups: endometriosis ; polycystic ovary syndrome ; age-related reduced ovarian reserve ; reduced ovarian reserve ; unexplained infertility ; genetic infertility . In particular, patients were assigned to the PCOS subgroup according to the Rotterdam 2003 diagnostic criteria. The diagnosis of PCOS was confirmed when two of the following three criteria were included: (1) menstrual irregularities typical of ovulatory dysfunctions, mainly with oligomenorrhea or amenorrhea; (2) clinical hyperandrogenism, such as hirsutism, acne, alopecia, and/or biochemical hyperandrogenism, detected by laboratory hormone assays; (3) polycystic appearance of the ovary on ultrasound examination. Differently, patients were assigned to the EM subgroup when, on anamnesis, some of the following symptoms were noticed: dysmenorrhea, dyspareunia, chronic pelvic pain, menorrhagia, dysuria, and/or hematuria, and dyschezia and/or rectal bleeding. On physical examination, EM was evidenced by pain in the bimanual examination and fixed or not very mobile uterus (when this disease is profound and involves a massive formation of fibrotic processes). Laboratory investigations to support the diagnosis of EM included the dosage of the serum CA-125 marker, transvaginal ultrasound , Nuclear Magnetic Resonance (to detect endometriotic foci in different locations of the pelvis). The combination of the results of these examinations/analyses strongly supported the diagnosis of EM, which was definitively performed after the histological examination of the biopsy of the endometriotic lesions. Furthermore, patients were assigned to the GI subgroup in the case of chromosomal aberrations, as well as in the case of mutations on genes known to cause infertility in females .Patients included in the study were recruited at the Alma Res Fertility Centre  from September 2018 to January 2020, with the approval of the Alma Res Ethical Committee . The groups of infertile females  (GnRH antagonist protocol). In EM, AR-ROR, and ROR, ovarian stimulation was induced by ultrashort flare GnRH analog starting on day 1 after the beginning of the cycle, followed by menotropin administration on day 2 after the beginning of the cycle, and by administration of r-HCG (ultrashort protocol). Patients underwent conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), depending on the male factor and the type of infertility. Conventional IVF was applied in the case of unexplained infertility and endometriosis, whilst ICSI was performed in the case of earlier IVF failure or suboptimal fertilization, i.e., when the fertilization rate was lower than 20%, as well as in the case of male infertility with severe sperm abnormalities.To reduce confounding factors, all participants (including controls) were interviewed to assess they had similar dietary patterns and lifestyles . Additionally, none of them was taking oral adjuvant/nutraceutical supplements during the 3 months before the analysis of their FF samples.g at room temperature and two aliquots of the resulting supernatants (500 μL each) were immediately processed for the analysis of metabolites, as subsequently indicated. Samples showing any visible trace of blood contamination were discarded.When at least three follicles reached an approximate size of ≥18 mm each, they were aspirated 36 h after the injection of r-HCG. Transvaginal, ultrasound-assisted follicles aspiration was performed with the vacuum pump. FF samples from the same donor were pooled, centrifuged for 10 min at 1500× 2, 6% CO2, 89% N2 at 37 °C. At 5 days post-insemination, the number of developed blastocysts was counted and the quality of blastocysts was evaluated, in accordance with the Gardner blastocyst grading system. Blastocysts were then assessed for transfer or vitrification. Oocytes, denuded of surrounding cumulus cells by treatment with hyaluronidase , were inseminated via ICSI 2 h later, while oocytes via IVF were inseminated, without denudation, 4 h after oocyte retrieval. Injected oocytes were transferred to a culture dish containing 50 μL global total LP medium overlaid with 10 mL Ovoil  in a pre-equilibrated GPS-Embryo culture dish , and then cultured under an atmosphere containing 5% OFor each patient, the number of retrieved oocytes, of mature oocytes, fertilized oocytes, blastocysts, high-quality blastocysts (defined as those scoring a grade ≥ 4AA), and positivity for β-HCG  were considered. In those patients who decided on the embryo transfer, the number of clinical pregnancies and of healthy offspring were also considered.The targeted metabolomic evaluation of FF samples was carried out by the Redox Energy Test , empowerg for 15 min at 4 °C to precipitate proteins and the clear supernatants were saved at −80 °C until the HPLC analysis of fat-soluble vitamins and antioxidants.A second 500 μL FF aliquot was light-protected and then processed to extract fat-soluble antioxidants using a method recently described in detail elsewhere . BrieflyIn all FF samples, the following water-soluble compounds related to purines and pyrimidines metabolism, energy metabolism, antioxidants, and oxidative/nitrosative stress were separated and quantified by HPLC according to methods set up in our laboratory ,51: cytoall trans-retinoic acid, all trans-retinol (vitamin A), 25-OH-cholecalciferol (vitamin D3), α-tocopherol (vitamin E), γ-tocopherol, δ-tocopherol, coenzyme Q10, total carotenoids .In all organic solvent extracts of FF samples, the following fat-soluble vitamins and antioxidants were separated and quantified by HPLC ,52: all ® software (version 5.0) package provided by the HPLC manufacturer.The Surveyor HPLC apparatus  was equipped with a highly sensitive 5 cm light-path flow cell diode array detector and set up for acquisition between 200 and 550 nm wavelengths. Water-soluble compounds, free amino acids, and amino group-containing compounds were loaded  onto a Hypersil C-18, 250 × 4.6 mm, 5 μm particle size column , while fat-soluble compounds (200 μL) were loaded onto a Hypersil Gold C-18, 200 mm × 4.6 mm, 5 μm particle size column . Both columns used were provided with their own guard columns. Data acquisition and analysis were performed using the ChromQuest® 6000 instrumentation . The amount of lactate in these FF extracts was determined spectrophotometrically using the method described by Artiss et al. [In all aqueous, protein-free FF extracts the concentration of glucose and ammonia were measured by automated procedures using the Cobass et al. ,54.p and q values of less than 0.05 were considered statistically significant.Statistical analysis was performed by using the GraphPad Prism program, release 8.0. Normality of distribution was tested by the Kolmogorov-Smirnov test. For all continuous variables, mean ± SD, median, minimum, maximum, quartiles (1st and 3rd), and percentiles (10% and 90%) were calculated, while frequencies were determined for categorical variables. Differences among the two groups of control subjects (CTRL) and infertile females (IF) were displayed by using the Mann-Whitney U-test and differences among CTRL and the groups of IF categorized into the 6 subgroups according to the clinical diagnosis  were assessed by the 1-way ANOVA, followed by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli for multiple comparisons, as the post hoc test to control the false discovery rate. A n = 27) and therefore being potentially considered as a biomarker for female infertility, we determined the 10% and 90% percentile of its concentration in each follicular fluid sample of the CTRL group. Subsequently, in the entire cohort of FF donors (CTRL and IF) each metabolite was assigned to one of the following two categories: (1) Normal (0) = when the metabolite concentration was ≥10% or ≤ 90% percentile of the CTRL group; (2) Positive (1) = when the metabolite concentration was outside the 10% or 90% percentile of the CTRL group. Subsequently, the concentrations of the various metabolites determined in the FF sample of each participant (of both CTRL and IF groups) were simplified into a specific profile of 0  and 1 (Positive), lastly generating a “Biomarker Score” (sum of the number of Positive in each subject). The Biomarker Score, therefore, ranged from 0, when a subject scored 0  for each of the metabolites identified as potential biomarkers, to 27 when a subject scored 1 (Positive) for each of the metabolites identified as potential biomarkers [For each of the metabolites showing significant differences between CTRL and IF (omarkers . It is wIn the preliminary analysis of the data, it was determined that the Biomarker Score values showed a normal distribution in both CTRL and IF groups. These normal distributions allowed calculation of the posterior probability curves using the Bayes theorem , thus peTo assess the correlation with the clinical parameters of fertility, subjects of the two groups were combined into a single group of FF donors and the value of the Biomarker Score of each subject was plotted as a function of the number of retrieved oocytes, of mature oocytes, of fertilized oocytes, of blastocysts, and of high-quality blastocysts. Linear correlations were calculated using Spearman’s correlation coefficient.


NLC-HG4%) displayed desirable rheological, mechanical, and mucoadhesive properties for topical application in the nasal cavity. Compared to control formulations, the use of TTCNLC-HG4% slowed in vitro permeation of the anesthetic across the nasal mucosa, maintained cytotoxicity against neuroblastoma cells, and provided a three-fold increase in analgesia duration, as observed using the tail-flick test in mice. The results obtained here open up perspectives for future clinical evaluation of the thermoreversible hybrid hydrogel, which contains TTC-loaded NLC, with the aim of creating an effective, topical, intranasal, needle-free anesthesia for use in dentistry.Recent advances have been reported for needle-free local anesthesia in maxillary teeth by administering a nasal spray of tetracaine (TTC) and oxymetazoline, without causing pain, fear, and stress. This work aimed to assess whether a TTC-loaded hybrid system could reduce cytotoxicity, promote sustained permeation, and increase the anesthetic efficacy of TTC for safe, effective, painless, and prolonged analgesia of the maxillary teeth in dental procedures. The hybrid system based on TTC (4%) encapsulated in nanostructured lipid carriers (NLC) and incorporated into a thermoreversible hydrogel of poloxamer 407 (TTC Similar process  and poly process  in assocl mucosa ,36.NLC-HG4% to be topically administered at the nasal mucosa and, possibly, to resist the shear stress caused by mucociliary activity, resulting in a longer residence time and enhanced drug absorption [Frequency sweep analysis revealed viscoelastic behavior, with the predominance of G’ over G”, at 32.5 °C, for all the formulations, as shown in sorption .HG4%.TPA is an essential tool to investigate the effects of different compositions of formulations on their mechanical properties ,39. Thisp > 0.05) between TTCNLC-HG4% and TTCHG4% in terms of the cohesiveness and elasticity parameters. On the other hand, TTCNLC-HG4% was significantly (p < 0.01) more adhesive (1.062 ± 0.056 N) than TTCHG4% (0.534 ± 0.154 N).As shown in Elasticity is a mechanical property related to the reversible deformation of a formulation submitted to compression stress, following a wait time between compressions, while cohesiveness indicates how well the formulation tolerates a second deformation, relative to its resistance under the first deformation ,40. It cNLC-HG, since the value of the phase angle (tan δ = G”/G’) of TTCHG was slightly higher than that of TTCNLC-HG. When tan δ becomes higher, the elasticity (solid character) of the formulation decreases, while the viscous behavior (liquid character) increases [NLC-HG and the nasal mucosa, leading to better clinical performance [In the case of adhesiveness, the value can be affected by the hydrogel viscosity. This could explain the higher adhesiveness of TTCncreases . In addiformance ,43.In the pharmacotechnical field, mucoadhesion can be defined as the interaction between a formulation and the mucin layer. This interaction is firstly achieved by contact, spreading, and swelling of the formulation on the mucosa, followed by consolidation involving chemical or physical bonds between the molecules of the formulation and the mucin ,45. In tTexture analyzers are commonly used to measure the detachment of hydrogels from artificial mucin ,40,44,47NLC-HG4% and TTCHG4%, as shown in The most commonly evaluated mucoadhesion parameters are (i) the maximum force necessary to separate the probe from the tissue (maximum detachment force), and (ii) the total amount of force involved in removing the probe from the tissue (mucoadhesion work) ,49. TherNLC-HG4%.The incorporation of TTC-loaded NLC into HG did not change the force required to detach it from the nasal mucosa. The presence of NLC in the hybrid hydrogel could not interfere with the flexibility of the polymer chain, due to its high density of crosslinks. Polymer chains with high flexibility can provide greater interfacial contact and a more favorable environment for entanglement between the polymer and the mucin molecules ,37,47, fHG4% was higher than that of the hybrid system, with TTCHG4% taking longer to complete its detachment from the mucosa during the probe withdrawal step. Since the detachment force was similar for the two formulations, the results suggested that the high mucoadhesion work of TTCHG4% could have been related to the low internal resistance of its molecules for withstanding external forces without breaking. Thus, the intermolecular bond strength was weaker for TTCHG4% compared to the TTCNLC-HG4% formulation, resulting in the better mucoadhesive performance of the latter [Interestingly, the mucoadhesion work of TTCe latter ,50.NLC-HG4%. Interestingly, the study also revealed that this formulation showed faster in vivo absorption of zidovudine when the drug was administered topically through the nasal mucosa compared to intravenous administration. This was suggested to be due to the anatomical, physiological, and histological characteristics of the nasal cavity, which provided mucoadhesion and, consequently, rapid absorption and onset of action [Carvalho et al.  reportedf action . An implHG4% and TTCNLC-HG4% across the porcine nasal mucosa, while ssJ) and lag time values calculated from the curves.p > 0.05). However, the linear regression analysis showed that permeation across the nasal epithelium mucosa was significantly (p < 0.0001) higher for TTCHG4%, compared to TTCNLC-HG4%. Hence, the encapsulation of TTC in NLC did not influence the initial permeation of TTC, but the permeation could be sustained for longer, and with a lower flux. Similar results were reported previously for the fluxes of local anesthetics encapsulated in polymeric nanocapsules, which were also significantly lower than obtained for the same anesthetics incorporated in a commercial formulation [There was no statistical difference between the formulations in terms of the steady-state flux and lag time parameters , decreasing the number of free anesthetic molecules available to cross the epithelial barrier, while at the same time prolonging the anesthetic effect ,52.HG4%, and TTCNLC-HG4%) for 24 h are shown in Despite the limitations of monolayer in vitro cell culture models, which can hinder correlation to in vivo studies, these are well-defined methods for evaluation of the potential cytotoxicity of drugs . The SH-p < 0.05) for TTCfree at 4 × 10−5 mg, compared to the other formulations (TTCHG4% and TTCNLC-HG4%). Nonlinear regression analysis was used to calculate the IC50 values for the TTC formulations (p > 0.05), which were 0.006, 0.008, and 0.01 mg·mL−1 for TTCNLC-HG4%, TTCHG4%, and TTCfree, respectively.The results only revealed a statistical difference  compared to TTCNLC-HG2% (26 h), TTCHG4% (10 h), and TTCHG2% (8 h). As expected, the formulations without TTC (HG and HG+NLC) presented no analgesia effect  across the skin found slower permeation, while the anesthetic effect in mice was prolonged .It should be noted that this antinociceptive test was performed with the application of the formulations on the skin of the tails of the animals. Many studies have observed that drugs permeate more easily across mucosas , compared to skin , due to NLC-HG4% administered by the intranasal route.The nasal mucosa is composed of squamous keratinized epithelium with a single layer of pseudostratified ciliated columnar respiratory cells, goblet cells, and the submucosal glands . The lowNLC-HG4%, compared to the drier skin surface. Hence, the anesthetic effect of TTCNLC-HG4% is enhanced by use of the intranasal route, since it is known that mucoadhesion can favor the rapid onset action of drugs [In addition, the moisture present in the mucus-covered nasal surface favors the process of mucoadhesion of TTCof drugs .NLC-HG4% in humans, given that previous studies have observed that the flux of an anesthetic can be used to predict the efficacy of an anesthetic in the preclinical phase [Therefore, the findings of this work open up perspectives for the excellent anesthetic performance of TTCal phase ,64.For the formulations without TTC (PL407 hydrogel (HG) and NLC incorporated in HG (HG + NLC)), the thermal sensitivity of the animals changed abruptly in the first 60 min of the tail-flick test, as shown in NLC-HG is a genuine candidate as a drug delivery formulation for use via the nasal route. Although an intranasal spray composed of 3% tetracaine plus 0.05% oxymetazoline already exists, its indication is limited to single maxilla restorative procedures in patients weighing at least 40 kg. In this case, the failure of TTC to anesthetize the posterior superior alveolar nerve was probably due to its inability to penetrate the more posterior regions of the maxillary sinus, compromising anesthesia in the region of the second premolar, with complete failure in the region of the first molar [NLC-HG4% can prolong both TTC permeation and the duration of anesthesia, which should encourage its clinical evaluation after topical nasal administration, aiming at providing effective analgesia that allows more invasive dental procedures, such as multiple tooth restorations, simple extractions, and endodontic procedures.Overall, the results obtained here were very encouraging, showing that TTCFrom a different perspective, the drug delivery system designed in this work could also be used as a nose–brain delivery system for the treatment of diseases such as migraine, cluster headache, and trigeminal neuralgia, for which the nasal route is preferable ,70,71,72NLC-HG hybrid formulation showed improved mechanical, rheological, and mucoadhesive properties, together with low cytotoxicity, sustained in vitro permeation, and extended in vivo anesthesia, compared to tetracaine directly loaded in the hydrogel. These findings support its clinical evaluation in order to establish whether it is able to provide effective, topical, intranasal, needle-free anesthesia in dentistry.TTC-loaded NLC were successfully incorporated in poloxamer PL407 hydrogel, with the formulation presenting desirable characteristics for the provision of topical, intranasal, needle-free anesthesia. The 4% TTC


ALP), bone morphogenic protein (BMP)2, collagen (COL)1A1, osteonectin (SPARC), osteoprotegerin (OPG), osterix (OSX), receptor activator of NF-κB (RANK), RANK Ligand (RANKL), and Runt-related transcription factor (RUNX)2. There were small differences in cell proliferation and LDH release regarding treatment duration and distance to the cells. However, an increase in the expression of RANK and RANKL was observed at longer treatment times. Additionally, CAP caused a significant increase in mRNA expression of genes relevant to osteogenesis. In conclusion, CAP has a stimulating effect on osteoblasts and may thus represent a potential therapeutic approach in the regeneration of hard tissue defects.Bone regeneration after oral and maxillofacial surgery is a long-term process, which involves various mechanisms. Recently, cold atmospheric plasma (CAP) has become known to accelerate wound healing and have an antimicrobial effect. Since the use of CAP in dentistry is not yet established, the aim of the present study was to investigate the effect of CAP on human calvaria osteoblasts (HCO). HCO were treated with CAP for different durations of time and distances to the cells. Cell proliferation was determined by MTT assay and cell toxicity by LDH assay. Additionally, RT-qPCR was used to investigate effects on osteogenic markers, such as alkaline phosphatase ( BMP2), Runt-related transcription factor 2 (RUNX2), osterix (OSX), and osteoprotegerin (OPG), and followed by markers that appeared later in osteogenic and mineralization cascade, as alkaline phosphatase (ALP), osteonectin (SPARC), and collagen 1 (COL1A1) [After oral and maxillofacial surgery, the healing of hard tissue is a much more complex and long-term process as compared to soft tissue. The regeneration of bone is particularly extensive and involves many different mechanisms, such as the differentiation of osteoblasts or the formation of collagen fibres and its arrangement according to the mechanical forces ,2,3. The(COL1A1) ,9,10,11.(COL1A1) ,13. Howe(COL1A1) ,15,16.Recently, cold atmospheric pressure plasma (CAP) has become known to improve wound healing ,18. CAP Although the detailed effects of CAP have not yet been fully elucidated, it seems to cause a highly regulated wound healing–promoting mechanism in various cells and tissues. Thus, among other aspects, stimulating effects on, e.g., keratinocytes, fibroblasts, or endothelial cells have been observed, in particular an upregulation of certain genes and proteins, an increase in cell proliferation, and higher cell viability ,23,24. FOral and maxillofacial surgery in particular could benefit from clinical use of CAP with the possibility of increased bone regeneration or the application of the anticancer effect. Promising studies with preosteogenic mice cell lines and human periodontal ligament cells showed an enhanced osteogenic differentiation potential by CAP ,30,31. IFirst, we investigated the effect of different treatment lengths and effects of remaining culturing medium during treatment on the cells. As shown in When an application is performed on patients, the continuous movement of the patient and the doctor causes the distance to change continuously. As shown in Next, we examined the effect of a single 60 s treatment versus multiple 60 s treatments distributed over several days, using an MTT assay. The CAP treatment resulted in an increase in cell viability only after about 10 d, both at a distance of 0.1 cm and at a distance of 0.5 cm. There was a clear difference between single and multiple treatments, especially after 10 and 14 d. Three treatments seem to have a negative effect on proliferation. Overall, cell proliferation was less pronounced at a greater distance between the CAP device and the cells a,b.RANK and RANKL, were analysed additionally .® GmbH, Nassau, Germany) as previously described [A dielectric barrier discharge was used to create ambient air generated CAP at an output of 18 kV  were used. Conditions for cell culturing were described previously .2 and allowed to adhere for 24 h. Various cell culture plates  were used. For the direct treatment, the culturing medium was removed immediately before the CAP application and was added freshly afterwards. For the indirect treatment, CAP was applied, leaving the cell culture medium on the cells. The distance indicated in the corresponding experiments was measured from the bottom of the dish and the CAP device was not in contact with the culturing medium. In the preliminary study part, various distances and impulse length were used to estimate nontoxic conditions. Osteogenic culturing medium was used [2 and allowed to adhere for 24 h followed by a CAP treatment with two distances (0.1 and 0.5 cm). In addition, cells for the control group were cultivated without CAP treatment. Samples were analysed 4, 10, and 14 days after the CAP treatment. The cell culture part was repeated three times.Cells were seeded with a density of 10,000 cells/cmwas used ,67. In twas used . Cells wThe proliferation rate was estimated with an in-house MTT assay and cytotoxic effects were determined with the Pierce™ LDH Cytotoxicity Assay  described before . The quaRUNX2, BMP2, ALP, TNFRSF11B, COL1A1, SPARC, SPP1, BGLAP, RANK, RANKL was analysed.RNA isolation, cDNA transcription, and real-time PCR analysis were described previously ,67. Specp < 0.05.The experiments were performed in triplicates and repeated at least twice by calculating mean values and 95% confidence interval. For statistics, data were log2 transformed. The statistical analysis was performed using GraphPad Prism Software . One-way ANOVA with post hoc Tukey’s multiple comparison test was used for CAP has a mediocre influence on differentiation potential of human osteoblasts. Cell viability is affected by the length of CAP treatment and by the distance of the CAP device to cells. Negative effects increased with longer durations of CAP treatment, with repetitions of CAP, and with larger distances to the cells. Nevertheless, CAP has positive effects on osteogenic differentiation potential. Regardless of shorter distances (0.1 or 0.5 cm), osteogenic marker genes are stimulated by CAP. Considering the bedside situation, these are negligible temporally delays in expression.


The analysis of differentially expressed genes (DEGs) indicated that 79 unique genes were dysregulated (39 upregulated and 44 downregulated genes). The results showed that MeHg altered gene expression patterns as a response to the cellular stress produced, reflected in cell cycle regulation, lysosomal activity, autophagy, calcium regulation, mitochondrial regulation, apoptosis, and regulation of transcription and translation. The analysis of DEGs showed a low response of the antioxidant machinery to MeHg, evidenced by the fact that genes of early response to oxidative stress were not dysregulated. The RBCs maintained a constitutive expression of proteins that represented a good part of the defense against reactive oxygen species (ROS) induced by MeHg.To understand changes in enzyme activity and gene expression as biomarkers of exposure to methylmercury, we exposed loggerhead turtle erythrocytes (RBCs) to concentrations of 0, 1, and 5 mg L Mercury (Hg) represents one of the most serious environmental threats to wildlife well-being ,2. It isIn the sea, MeHg is a molecule of great concern to marine populations as it bioaccumulates and biomagnifies in tissues, causing health damage . MeHg maMeHg is a weak electrophile with a strong affinity for sulfhydryl groups (-SH) and selenoles (-SeH), which are functional in proteins containing cysteines and selenocysteines respectively, with consequences such as (i) inactivation of molecules related to electrophilic forms, (ii) decrease in Se availability and, therefore, selenoprotein synthesis, and (iii) increase in intracellular MeHg concentration ,9.Exposure to MeHg has been extensively studied and is well documented in mammals, birds, and fish and includes neurotoxicity, impaired growth and development, reduced reproductive success, liver and kidney damage, and immunomodulation ,12,13,14Advances in next-generation sequencing technologies, such as massive RNA sequencing (RNA-seq), allow for the characterization of molecular responses to stimuli with impressive sensitivity for non-model organisms, such as the loggerhead turtle ,23,24. Twww.iucnredlist.org, accessed on 13 January 2021). For this reason, it is important to propose alternative study methodologies that do not compromise the life of these animals that are instrumental for the health of marine ecosystems [Studies carried out on the toxicity of MeHg in wild animals and humans using next-generation sequencing (RNA-seq) have generated knowledge about the molecular mechanisms of this compound produced in different tissues ,29,30,31osystems . Recentlosystems . These cosystems ,35,36,37osystems .−1 of MeHg for 12 h. The observed response shows the increase in reactive oxygen species in RBCs [Caretta caretta, to identify cellular pathways or biological processes affected by MeHg while preliminarily elucidating the molecular mechanisms of toxicity used by this xenobiotic against this marine turtle.In previous studies, we demonstrated that an increase in the enzymatic activity of Glutathione S transferase and superoxide dismutase, as well as the production of malonaldehyde, was revealed when loggerhead turtle RBCs were exposed to 0, 1, and 5 mg L in RBCs . In thisThe samples and methods used in this study followed the ethical standards of Colombian legislation and those of the ethics committee of the Jorge Tadeo Lozano University (UJTL) (Project 340-07-10). The project had a permit to collect samples of Colombian biodiversity, granted to the UJTL by the Ministry of the Environment .Peripheral blood was collected from five captive, pre-juvenile loggerhead turtles at the El Rodadero Aquarium in Santa Marta . The details of blood sample collection and RBC isolation have been described previously . In brie6 RBCs/ml from five peripheral blood samples were isolated by centrifugation and then resuspended in 1.5 mL Eppendorf tubes in MEM-S medium [−1 of methylmercury II chloride (CH3HgCl)  for 12 h at 30 °C. Each of the five samples was exposed to the three concentrations of CH3HgCl (MeHg), thus producing 15 samples altogether. As a control, we used RBCs in a culture medium with sterile saline solution (used to dilute the MeHgCl) (Aliquots of 2 × 10S medium . We expo MeHgCl) .® , and stored at 4 °C for one week.At 12 h of incubation, the RBCs exposed to MeHg were washed twice with 0.9% sterile saline solution , then centrifuged at 2500 rpm at 22 °C, resuspended in 15 volumes of RNAlaterg for 10 min at 22 °C. We extracted total RNA from each of the samples (~25 µL RBCs/mL) using RNeasy® Mini Extraction Kit, following the instructions of the manufacturer . The extracted RNAs were treated with DNase according to the instructions of the manufacturer   and diluted in RNAsa-free MilliQ water. RNA was stored at −80 °C for subsequent evaluations.Before extraction, the RNAlater was removed and the RBCs were washed with 1 volume of 0.9% sterile saline solution (4 °C), then centrifuged at 5000× The RNA was quantified and its quality analyzed by spectrophotometry, using a Nanodrop-1000 . The RNA was evaluated by electrophoresis in 1% agarose gels . Each of the total RNA preparations was individually assessed for RNA quality based on the 28S/18S ratio and the RNA integrity number (RIN) was measured by 4200 TapeStation capillary electrophoreses  at the Research Center of the Colombian Agricultural Research Corporation in Tibaitatá, Cundinamarca . Samples with RIN > 8.5 were selected for further analysis . The Bei−1 = Gs1 and 5 mg L−1 = Gs5), and the union of all samples, using default parameters [We evaluated the raw sequencing data using the FastQC tool . Then, trameters . We evalrameters , compari−5) [We grouped the obtained transcripts in pairs according to the similarity of the sequences to produce longer and more complete consensus sequences using the TGICL v2.0.6 program (a software system for fast clustering of large EST datasets) . The res−5) . Later, −5) . We used−5)  to deter−5) . Unigene−5) .p value ≤ 0.05), which normalizes the data taking into account the differences in the total number of reads per library. Further, we used PossionDis . We mapped the clean reads to the unigenes obtained previously using Bowtie2 v2.2.5  and calcThe DEGs were identified by comparing the control group (Gc) with each of the groups exposed to MeHg (Gs1 and Gs5), and comparing the two exposed groups with each other (Gs1 versus Gs5). Subsequently, the distribution of the expressed genes was graphed using heat maps and MA plots with the heatmap and ggmaplot (DEseq2) functions, respectively. In the heat maps, we divided the expression levels of the DEGs into orange and blue regions that indicated high and low levels of relative expression, respectively. We compared the set of differentially expressed genes shared between the different treatments with Venn diagrams using the Venny 2.0 program .Differentially expressed genes were mapped to GO terms and the KEGG database to perform functional enrichment and metabolic pathway analyses, respectively. We then calculated the false discovery rate for each p-value. Terms whose FDR was less than 0.01 were defined as having significant enrichment.In a previous experiment, we used the same RBC samples  as in thAs dependent variables in each of the analyses, the following were selected: GST activity, Cu/Zinc-SOD activity, Mn-SOD, and MDA concentration (µM), respectively. As independent variables, the following were selected: relative expression ), the initial total concentration of MeHg, and concentration of MeHg added to each individual. The Tbxas1 gene was correlated with the concentration of MDA because this gene participates in the isomerization of the PGH2 prostaglandin, transforming it into 12-hydroxy-5, 8, 10-heptatrienic acid (12-HHT), and malondialdehyde (MDA).All data are available at NCBI under project accession number PRJNA575050. The raw sequences data of RNA-seq in FASTQ format were deposited at the NCBI in the Sequence Read Archive Database (SRA) under the accession numbers: SRR10412101, SRR10412100, SRR10412099, SRR10412098, SRR10412097, SRR10412096, SRR10412095, SRR10412094, SRR10412093, SRR10412092, SRR1041209, and SRR10412090.The sequences of the 12 libraries presented high quality according to the FASTQC analysis . However, sequencing reads containing adapter contamination, and unknown bases (N), were removed  . We asseThe de novo assembly of the twelve individuals produced a composite transcriptome with 121,933 unigenes, with an average length of 1444 bp, N50 of 3520, and a GC percentage of 46.98% . The sizChelonia mydas, Pelodiscus sinensis, and Chrysemys picta belli), and against a previously published Caretta caretta transcriptome (Genbank PRJNA560561), obtaining significant agreement against 97,546 proteins (80.5%) and 110,846 unigenes (90.9%), respectively.We generated unigenes for each of the assembled transcriptomes. The composite transcriptome annotation produced 72,700 (59.62%) unigenes with significant agreement against at least one of the analyzed databases . A totalTo evaluate the expression characteristics of the mRNA, we estimated the relative expression levels (FPKM) of each of the samples for each transcript . We clasp > 0.05). It is important to note that when Gs1 was compared to Gs5, it showed a marked reduction in gene expression  in all three categories (−1) generally decrease gene expression while higher concentrations increase it (5 mg L−1).On average, each of the 12 samples expressed 80,453 genes. No significant differences were found between the number of genes expressed between Gc, Gs1, and Gs5  between the Gc–Gs1, Gc–Gs5, and Gs1–Gs5 treatments. The group of upregulated genes under MeHg stress produced 39 matches and the downregulated genes produced 44 matches. These differentially expressed genes were annotated to the databases as follows: Nt , Nr , SwissProt , KEGG , KOG , InterPro  and GO . In summary, only 80 genes produced significant matches and were annotated to at least one database. The other three genes had no matches and could not be annotated (NC). Of the 83 dysregulated genes, four genes were in more than one comparison. This means we identified a total of 79 unique DEGs .When the gene expression of the Gc and Gs1 treatments were compared, we found 16 upregulated genes (between 1 and 7 fold) and 24 downregulated genes (between −1.7 and −5 fold) A,B Tabl. The comWe found that none of the upregulated and downregulated genes were common to all three established relationships . Of the The enrichment of GO terms of DEGs was carried out to identify the related biological processes and the functions affected by MeHg in the RBCs. The GOs were assigned to the three gene ontologies: biological processes, cellular components, and molecular function. Of the 79 DEGs, only 27 were annotated against the GO database (11 upregulated genes and 16 downregulated genes). When Gc was compared with Gs1, 113 unigenes were classified into 28 GO functional group categories . When comparing Gs1 with Gs5, 103 unigenes were classified into 27 GO functional group categories , and finally, when comparing Gc with Gs5, 17 unigenes were classified into 17 GO functional group categories  .To acquire an understanding of what may be happening at the cellular level between Gc, Gs1, and Gs5, the annotation was completed manually using the Uniprot and Gene Ontology databases. The differentially expressed unigenes in the three comparisons were classified according to molecular function in the following categories: oxidative stress, regulation of the cell cycle, signaling, regulation of transcription and translation, binding to metal ions, autophagy, apoptotic processes, metabolic processes, membrane transport, DNA repair, mitochondria, and miscellaneous. The results of the top 25 annotations are presented in The DEGs between Gc and Gs1 presented 40 dysregulated unigenes which were grouped into 11 GO categories according to their function. The most representative categories were: regulation of transcription (two upregulated and ten downregulated genes), signaling (one upregulated and seven downregulated genes), oxidative stress (three upregulated and three downregulated genes), metabolic processes (three upregulated and two downregulated genes), and binding to metal ions (four upregulated genes) . The DEGThe enrichment analysis of KEGG pathways carried out on differentially expressed genes showed that, when comparing Gc to Gs1 A, Gs1 toR = 0.92, 0.75, 0.94 and 0.89, respectively) (p < 0.001) (R = 0.75 (p = 0.005) and R = 0.67 (p = 0.016), respectively  and the concentration of MDA were directly correlated with the relative expression of the Cu/Zinc-SOD, Mn-SOD, GST, and Tbxas1 genes (< 0.001) A–D. Addiectively .Chelonia mydas, Pelodiscus sinensis, and Chrysemys Picta belli) have already been published, and there are 37 turtle genomes in the process of assembly, 10 of which also have been published, representing important progress for the understanding of these chelonians.The results of our study provide information on the differential expression of genes in loggerhead sea turtle RBCs exposed to MeHg. It should be noted that the information available on non-model organisms is scarce and, therefore, requires development. We highlight that only 59% of the unigenes were annotated in at least one of the seven databases used, and of the 79 unique DEGs identified, only 27 were annotated against GO. This result shows the knowledge gap on the subject. Furthermore, several genes that were matched against databases were genes with unknown functions, and few studies have been conducted using transcriptomics to evaluate gene dysregulation caused by MeHg. Clearly, the lack of functional annotation of transcriptomes from non-model organisms is limiting the development of the mechanistic understanding of complex traits . HoweverFor these reasons, our study represents an important advance of DEGs analysis in the understanding of the toxicity mechanisms by which MeHg affects cellular homeostasis and of the adaptive response of loggerhead sea turtle RBCs. Adaptive responses to MeHg toxicity appear to involve complex polygenic processes. Our findings were consistent with this and reveal that several functionally distinct genes were dysregulated in response to stress produced by MeHg.In the comparisons made to determine the differential expression of genes between Gc–Gs1, Gs1–Gs5, and Gc–Gs5, the greatest dysregulation occurred in downregulated genes (44 genes) related to the response to cellular stress, signaling, transcription, calcium metabolism, and transport across the membrane, and in upregulated genes (39 genes) involved in the response to stress, lysosomes, mitochondria, regulation of the cell cycle, metabolic processes, and transcription and translation .These results demonstrate that RBCs in Gs1 generally expressed fewer genes, but their response to MeHg was more pronounced (40 dysregulated genes) than RBCs in Gs5 (12 dysregulated genes). The identification of changes in gene transcription can contribute to the identification of molecular events that are in the process of initiation or to the identification of the sequence of molecular events that can lead to cellular dysfunctions, as well as the identification of toxicity markers ,68. StudOxidative stress produced by an increase in reactive oxygen species is one of the earliest responses of RBCs to MeHg toxicity . In thisThe Sgk1 and MkNk1  genes weIn this study, we found that the expression of Atg5 shows that there is cellular damage in RBCs and its overexpression produces compensation. In mice, the silencing of Atg5 in cells exposed to acute oxidative stress has produced the overexpression of cytokines, which generate inflammation and cellular apoptosis . These fLikewise, we identified the overexpression of the GPD1 gene. The enzyme encoded by this gene reduces dihydroxy-acetone phosphate to glycerol-3-phosphate (G-3-P), reducing in turn one mole of NAD to NADH . Shen etAnother downregulated gene in the RBCs that is also related to oxidative stress is KPNA6, which is a transport adapter between the nucleus and the cytoplasm and is related to the regulation of cytokine production involved in the inflammatory response to stress . DysreguNrf2 activation is negatively regulated by Keap1, which exports the Nrf2 transcription factor from the nucleus to the cytoplasm ,82. The The antioxidant defense systems are essential to neutralize high levels of ROS, which can cause irreversible damage to cells. In this way, cells have antioxidant molecules and detoxifying enzymes that can control free radicals. GSH is the most abundant antioxidant small molecule. Detoxifying enzymes include SOD, GPx, glutathione-S-transferases (GSTs), CAT, glutathione reductase, glutamate-cysteine ligase (GCL), NAD (P) H: quinone oxidoreductase (NQO1), heme oxygenase-1 (HO-1), and other phase II detoxifying enzymes ,13. ThesHeat shock proteins serve many functions as molecular chaperones and folding guide proteins and by preventing protein build-up. During events such as oxidative stress, the expression of heat shock proteins is known to increase considerably . In geneLow concentrations of MeHg induce autophagy , which iWe identified the following upregulated genes: HEX_A, which catalyzes the degradation of GM2 gangliosides to GM3 gangliosides in lysosomes , moleculWe found that the SLC38A9 gene, which plays a role as an amino acid sensor upstream of mTORC1 for asparagine, arginine, glutamine, histidine, and lysine, was downregulated . SLC38A9It is clear that the RBCs in the culture medium in which the bioassay was carried out did not have the necessary environmental and hormonal signals for growth. Thus, the activity of the SLC38A9 gene was decreased and its signaling mechanism remained inactive; therefore, RBCs did not reproduce, nor did mTORC1 activation occur . It is i−5 M of MeHg, the depolymerization of the microtubules began, reaching the total inhibition of polymerization at a concentration of 3 × 10−5 M MeHg. They also observed that 15 MeHg molecules had been attached to the 15 sulfhydryl groups that tubulin (proteins that makeup microtubules) has. This response is what determines the fragmentation of microtubules [The cytoskeleton is involved in cell movement and division and is one of the primary targets of MeHg inside the cell. Specifically, it fragments microtubules, disrupting networks that are necessary to perform important biological functions . Vogel eotubules .The stability of the cytoskeleton of cells is dependent on spectrins and RhoGTpases ,100. SpeWe evidenced the upregulation of the spectrin SPTAN1 and the ARHGAP20 RhoGTPase genes. It is highly probable that, as a primary toxicity effect, MeHg initiated an attack on the cytoskeleton of the RBCs. This fact affects important functions, such as the mobilization of secretion and excretion vesicles, displacement of organelles, and intracellular transport of substances, as well as cell division (mitosis and meiosis) ,102,103.Other genes that regulate the cell cycle were also upregulated in the RBCs. This was the case with CEP250, UHRF2, and CTBP1 . The CEPGiven that UHRF2-CTBP1 and p53 are all involved in cell cycle regulation, the hypothesis that this is a new signaling pathway in the cell must be studied ,110. PosRegarding the CEP250 gene, it showed a relatively low upregulation in the RBCs. Probably, this was due to the fact that the RBCs did not have conditions for their reproduction in our bioassay. This adaptive response against MeHg demonstrates cellular damage at this level.2+ [2+ concentration is regulated in RBCs because calcium is a very important element in metabolic and intracellular signaling regulation [2+ regulation, blocking of voltage-dependent Ca2+ and Na+ channels in the plasma membrane, ultrastructural changes, and accumulation of MeHg within mitochondria. At the in vitro level, MeHg inhibits several mitochondrial enzymes and depolarizes the mitochondrial membrane, subsequently reducing ATP production [One of the most widely documented effects produced by MeHg is related to glutamate-facilitated excitotoxicity, a mechanism that causes excess intracellular Ca2+ . The cytgulation . In primgulation . Atchisogulation  reportedoduction .2+ overload in the cytoplasm, mediated by excitatory amino acid receptors (N-methyl D-aspartate (NMDA) and non-NMDA types) and by Ca2+ channels [2+ is distributed between the mitochondria and the smooth endoplasmic reticulum (ER). Mitochondria show low affinity and high capacity to transport Ca2+, while the ER has high affinity and low capacity to transport Ca2+ [It has been established that in vitro cell exposure to MeHg generates a Cachannels ,115. Theort Ca2+ ,117.2+, when there is an imbalance increasing its intracellular concentration, initially produces pressure for the ER and the mitochondria to absorb part of the Ca2+ and avoid apoptosis. However, this triggers the fragmentation of the mitochondria via fission by MFF and the exit of cytochrome C from the mitochondria, which generates greater fission and apoptosis [The RBCs presented three dysregulated genes: MFF, ATP13A1, and MSTO1. MFF regulates the fission of mitochondria in association with Drp1 . Everythpoptosis . On the poptosis .The MSTO1 gene is also poorly studied. A mitochondrial location is suggested where mitochondrial morphology (via fusion) and distribution are regulated ,121. FisMeHg can cause DNA damage by oxidation and by its affinity with macromolecules . Some stC. elegans genome to low concentrations of MeHg and HgCl2 (1 mM), observing minor DNA damage; suddenly, however, a higher concentration of MeHg (5 mM) decreased the damage compared to controls. This result is in agreement with our findings in this study, where Gs1 expressed 11% fewer genes than Gs5. In the same way, we found evidence of the dysregulation of a good number of genes related to transcription and translation (22 and 4 genes respectively), with the downregulated genes being of greater quantity than the upregulated genes (15 and 11 genes respectively). It is important to note that the highest number of dysregulated genes was present in the Gc–Gs1 comparison (14 genes) and the lowest in the Gc–Gs5 comparison . Between Gs1 and Gs5, an intermediate number of genes (11 genes) were dysregulated.In an interesting study, Wyatt et al.  exposed Four dysregulated genes related to the regulation of transcription and gene silencing were detected . Three oIn contrast, the PIAS2 gene was upregulated. The enzyme that encodes the PIAS2 gene contains two structural motifs (finger ring and SUMO binding) and one domain (SAP domain) that activate or repress the Elk-1 transcription factor, which depends on the MAPK pathway ,129,130.p = 0.29 and Hb beta p = 0.24).At the same time, the transcription factor gene SOX6 participates in pre-mRNA splicing , stimula, and amphibians (7.2). Therefore, we hypothesize that, as loggerhead turtle hemoglobins are not very reactive, destabilization of their structure by mercuric ions is less possible.Ancora et al.  showed tWe infer that, in Gs1, MeHg alters the expression of the alpha hemoglobin in the RBCs due to the downregulation of the SOX6 gene, triggering the upregulation of the alpha and beta hemoglobin, resulting in a greater production of ROS and, thus, reducing the overall expression of genes. It is not clear why the SOX6 gene is upregulated in Gs5 and downregulated in Gs1 yet, the only difference between the two groups was the concentration of MeHg.MeHg is an electrophile that regularly attacks nucleophilic groups (thiols and selenoles) . Selenopp < 0.05) between Gc and Gs1, showing higher relative expression in Gc than in Gs5 and Gs1. SMS is a polyamine with antioxidant and anti-inflammatory properties that have been reported to significantly inhibit the production of nitric oxide (NO), prostaglandins, and cytosines [The analysis of DEGs carried out in this study found that the enzymes that represent the first line of the antioxidant machinery in response to MeHg were not dysregulated. We did not find dysregulation in genes that encode selenoproteins or thiol-proteins. However, to find out which genes were being expressed, we analyzed metabolic pathways, and we detected 397 metabolic pathways in which all unigenes participate. Among these, we found the metabolisms of cysteines and methionines, glutathione, selenocompounds, and peroxiredoxins. ytosines , and redPrynops hilarii was 1.9 ± 0.2 mM. This result provides grounds to think that the high content of -SH groups must be part of a redox buffer, an output antioxidant to counteract ROS and oxidative stress.In glutathione metabolism B, the rep < 0.05). Among the group of peroxiredoxins, PRDX2 is the most efficient enzyme in the elimination of ROS and H2O2, and the second most abundant protein in the RBCs after hemoglobin [Trachemys scripta elegans turtles and Chrysemys picta marginata hatchlings, identified genes that are constitutively upregulated in different organs of these turtles, including: mitochondrial genes encoding electron transport chain proteins, iron storage proteins, serine protease inhibitors, transmembrane solute carriers, receptor proteins of transport and neurotransmission, chaperone proteins, and, most importantly, antioxidant enzymes. These authors proposed “the maintenance of constitutive protection mechanisms”. Turtles maintain a constitutive expression of many proteins that represent the defense against oxidative stress, anoxia, aging, and disease. While expensive in terms of energy consumption, it is very important to provide immediate protection against any metabolic attack.In the metabolism of selenocompounds C, the remoglobin ,120, whemoglobin ,142. We moglobin , workingWe found a high statistical correlation between these variables . These aPerinereis nuntia), a correlation has been found between the GST gene relative expression levels and GST activity after exposure to a 50 µg L−1 dose of copper , which was given by the different GST isoforms (GST-omega and GST-sigma) [Perinereis nuntia individuals exposed to doses of 50 μg L−1 of Cd and found a high correlation between the relative expression levels of the GST-sigma and GST-omega isoforms and the total GST activity .In polychaetes (T-sigma) ,145. AnoT-sigma)  used PerDugesia japonica), exposed them to glyphosate and found a correlation between total SOD activity and the relative expression levels of the enzyme Cu/Zn-SOD (Pearson r = 0.62).Franco et al.  studied The results obtained in this correlation show a high relationship between the activity measurements made with classical biochemistry and the relative expression of the genes measured in FPKM. It is important to note that the comparison of more genes could give more robustness to this analysis.The toxicology of sea turtles is a relatively new field of research, yet the inclusion of RNAseq, together with bioinformatics and biochemical analyzes, represents a field of study for the present and future of toxicological research for these threatened and endangered animals. The threats posed by contamination by MeHg and other xenobiotics are imminent, and the rigorous study of their action mechanisms is imperative.We have identified DEGs that affected biological processes in loggerhead turtle RBCs by exposure to MeHg. The downregulated genes were related to cell stress response, signaling, transcription, calcium metabolism, and membrane transport. On the other hand, the upregulated genes were involved in response to stress, lysosomes, mitochondria, regulation of the cell cycle, metabolic processes, transcription, and translation.At low MeHg concentrations, in Gs1, gene dysregulation was higher compared to Gs5, producing a greater number of down- and upregulated genes. Furthermore, the total number of genes expressed in Gs1 was also lower (−11%) compared to RBCs exposed to a higher concentration of MeHg (Gs5). Everything seems to indicate that low concentrations of MeHg produce greater dysregulation. This hypothesis must be studied in depth to be corroborated.We found that the RBCs in Gs1 evidenced a greater expression of alpha and beta hemoglobin, which could be related to ROS generation at a higher rate than in Gs5, a fact that could also explain greater dysregulation and lower total gene expression.According to the analysis of DEGs, a low response of the antioxidant machinery of the early reaction to the toxicity of MeHg can be noted, evidenced by the fact that these genes were not dysregulated. Nonetheless, the expression analysis of the metabolism of cysteines and methionines, glutathione, selenocompounds, and peroxiredoxin enzymes showed a constitutive expression that could be related to “preparation for oxidative stress”, a theory proposed by the biochemist Evaldo Reischl , who argThe DEGs identified in this study provide a baseline for further studies on the impacts of MeHg oxidative stress on loggerhead turtle RBCs. We analyzed metabolic pathways using all identified unigenes and detected 397 metabolic pathways in which unigenes participate. This type of analysis has not been widely explored in Hg studies. Transcriptomics and the great advance of bioinformatics support the development of studies in ecotoxicogenomics, providing answers on the molecular mechanisms of toxicity used by MeHg against this sea turtle. There are no previous in vitro or in vivo studies on differential gene expression in RBCs or other tissues in loggerhead turtles. Therefore, it is important to develop additional studies to elucidate the transcriptomic responses of RBCs and other tissues of this turtle when exposed to MeHg.


Anionic surfactants (AS) are detrimental aquatic pollutants due to their well-characterized toxicity to aquatic organisms. The concentration of AS in aquatic environments is increasing because of their extensive use in many industries and households. The standard reference method for AS analysis is to determine a methylene blue active substance (MBAS) complex formed between AS and the methylene blue (MB) cation by using chloroform. However, chloroform has a low AS extraction efficiency and other limiting properties, such as a high density and volatility, which make the conventional AS analytical method time-consuming and labor-intensive. In an effort to replace the use of chloroform, this study was carried out to screen novel solvents for their ability to extract AS in water samples. Criteria were based on AS extraction efficiency, physicochemical properties, and the stability of the solvent under different environmental conditions. Organic solvents, such as methyl isobutyl ketone (MIBK), 1,2-dichloroethane (DCE), dichloromethane, benzene, and n-hexane, were assessed. In extraction of the anionic surfactant sodium dodecyl sulfate (SDS), the mixture of MIBK-DCE (3:1) proved to be an optimum solvent as an alternative to chloroform. It not only enhanced SDS extractability but also improved properties, such as having a lower volatility, a lower density than water, and a quicker phase separation. Among solvents screened, no one single solvent in SDS extraction could meet such criteria. The performance of the MIBK-DCE (3:1) mixture in SDS extraction was stable, irrespective of pH and ionic strength of the SDS solution, washing process, and presence of cations. Anionic interference from halogen and polyatomic and organic anions in SDS extraction by MIBK-DCE (3:1) existed only at an elevated concentration, which is not occurring in the natural aquatic environment. Results demonstrated that a MIBK-DCE (3:1) mixture solvent could be used in AS analysis for a wide range of aquatic samples and it could be the basis for the development of a new analytical method to replace conventional chloroform. Surfactants are considered to be one of the most undesirable contaminants in aquatic environments because of their well-characterized adverse impacts on aquatic organisms as well as terrestrial ecosystems ,4,5,6,7.Surfactants have amphiphilic characteristics and consist of a hydrophilic head and a hydrophobic tail component. They are categorized, depending on the charges of the hydrophilic head, into anionic, cationic, amphoteric, and nonionic forms ,9,10. ThAmong surfactants, anionic surfactants (AS) are used more frequently in care products and household cleaning products than other types of surfactants, because of their low cost, high foaming efficiency, and, thus, high washing efficiency even at a low temperature ,9,10,14.−1~mg L−1 ranges that, in many cases, exceed the regulatory level 11OSO3Na, 99.0%) was purchased from Wako Pure Chemicals  and used as the standard anionic surfactant. Solvents to compare the AS extraction efficiency in this study were chloroform , methyl isobutyl ketone , 1,2-dichloroethane , dichloromethane , benzene , and n-hexane . Methylene blue  and the above solvents were purchased from Wako Pure Chemicals , except for DCE and MIBK, which were obtained from Junsei Chemical  and Daejung Chemical , respectively. Concentration of MB as a cationic dye was prepared at 0.025% solution. All chemicals used were reagent grade.Sodium dodecyl sulfate (SDS: CH−1), in which each organic solvent (25 mL) was transferred to a previously dried and weighed aluminum dish and the dishes were weighed every 5 min for 2 h in an environment-controlled chamber. This measurement was triplicated.Physicochemical properties of the selected organic solvents, including density, health hazard, vapor pressure, and odor threshold, were surveyed from references. This study measured the volatilization rate , were added to each separatory funnel and shaken for 3 min. Afterward, phase-separation time was recorded. After decanting the majority of the water layer, the solvent layers were filtered through water repellent phase separating paper . The maximum absorbance of the extracted methylene blue active substance (MBAS) and corresponding wavelength were measured with a UV/Vis spectrophotometer . The extraction efficiency of each organic solvent was determined based on the concentration recovery (%) at the maximum absorption wavelength. All measurements were triplicated.The SDS standard solution was prepared at a concentration of 0.8 mg LTo screen novel solvents for SDS extraction, in an effort to replace the use of chloroform, the use of mixed solvents was investigated in addition to using a single solvent. With the use of a single solvent, it was impossible to meet the physicochemical properties of the extracting solvent that are required for optimum extraction of SDS in water samples. The optimum criteria for the screened solvent mixtures considered in this study were high SDS extraction efficiency, short phase-separation time between water and solvent layers, low volatility, and density lower than water. If density of a mixed solvent is lower than that of water, phase separation becomes much easier.Two of six solvents tested in this study were mixed as a factorial combination and their efficiencies were determined through preliminary tests . The optimum combination was selected based on the above-stated criteria. The screened solvents satisfying the above criteria were MIBK and DCE. To find the optimum mixing ratio of the two solvents, 10 different mixing ratios  were tested. Then, the SDS extraction efficiency and the above-mentioned criteria were compared with those of chloroform. All measurements were triplicated.® 1PS phase separator filter paper  without washing, and, in ‘B’ batch, the layer of extraction solvent was similarly filtered after washing it once with distilled water. The maximum absorbance and the corresponding wavelength of the extracted MBAS by each solvent were measured and compared. All measurements were triplicated.In the official reference method for determining AS, a washing process in the solvent layer after phase separation is required to remove impurities ,30,36. T−1) and 50 mL distilled water were transferred to a 100 mL volumetric flask. Solution pH was adjusted between 2~12 with dilute H2SO4 and NaOH, keeping the ionic strength at 0.32 M with 1 M Na2SO4. Final volume was adjusted to 100 mL with distilled water. After that, the 0.025% methylene blue solution (5 mL) and the selected MIBK-DCE mixture solvent (50 mL) were added to an aliquot of SDS standard solution and shaken for one min. After phase separation, the water layer was removed and the solvent layer was washed with distilled water and filtered through Whatman® 1PS phase separator filter paper . The absorbance of extracts was measured with a UV/Vis spectrophotometer  at 658 nm. All measurements were triplicated.Five mL of SDS solution  on SDS extractability by the mixed solvent, concentration of each of the respective cationic solutions was maintained at 0.1 M using Na2SO4, K2SO4, MgSO4, or (NH4)2SO4. The SDS standard solution and MIBK-DCE (3:1) solvent were mixed. The SDS extraction efficiency was determined using the same procedure as that for pH effects. Relative interference (%) by cations was assessed based on the recovery concentration of SDS in a cation-treated solution as compared to that of the control (100%). All measurements were triplicated.To test interference by cations ). This equation estimates the relative intensity of MBAS from the SDS and anion-treated solution (M) as compared to the intensity of MBAS from 1.0 mg L−1 SDS (3.5 × 10−6 M) which is set as 1. Thus, the higher the IS value is, the higher interference by anions.To test of interference by anions, solutions of NaF, NaCl, KBr, KI, NaNOAn ideal solvent for determination of SDS in water samples should have such properties as a high extractability, a short phase-separation time, a low volatility, a low solubility in water, a lower density than water, and a lower potential health hazard ,20,34,39−1) and the phase-separation time when the SDS was extracted by six different solvents. This study screened six solvents  to select which solvent is better than chloroform for SDS extraction. Extraction efficiencies and properties were compared. After compensating for the absorbance of the blank, the absorbances of SDS solutions extracted by 1,2-dichloroethane (DCE), MIBK, and dichloromethane were higher than the absorbance of the chloroform extraction, indicating higher SDS extractability . HoweverAs shown in Based on these results, DCE , MIBK, and dichloromethane were screened as candidates for further testing, even though DCE and dichloromethane had longer phase-separation times than chloroform. Physicochemical properties of the solvents  govern tIf the density of the solvent is greater than water, phase separation yields an extractant layer on the bottom and an aqueous layer on the top between the two phases. Consequently, the analytical process can be complicated and time-consuming due to the fact that a different separatory funnel is required for the washing process, which results in a longer time for analysis ,19,29,36If the extractant has a high volatility, the vapor should be removed frequently when the extractant is mixed with the sample, because it can increase the risk of human exposure and result in a loss of MBAS together with the chloroform vapor. Furthermore, since the extract volume could be reduced by volatilization during the analysis, the solvent volume must be made constant before absorbance measurement ,36. −1) lower than water. The vapor pressure of MIBK was the lowest at 20 °C, and the vapor pressure of DCE, chloroform, and dichloromethane were 4, 11, and 23 times higher than that of MIBK, respectively. The volatilization rates of MIBK, DCE, chloroform, and dichloromethane were 0.04, 0.12, 0.28, and 0.50 mL min−1, respectively. Judging from the boiling points, MIBK and DCE are heat stable. As far as safety is concerned, MIBK was considered as non-carcinogenic to human health, but other solvents were classified in the B2 class  to dimethylthionoline, which resulted in the formation of pink colors in a chloroform phase [rm phase . ResultsThe official reference method for AS analysis ,30 is knWhen cations and anions co-exist with AS in water samples, they compete with AS for MBAS complex formation, resulting in a colorless complex between AS and MB ,29,46. E2+, NH4+, K+, Na+) on SDS extractability by the MIBK-DCE (3:1) mixed solvent, as compared to that in distilled water. The extraction efficiency was slightly decreased (2~3%) when cation concentrations were 0.1 M, and this concentration is hardly detectable in natural surface water. Magnitude of interference by Mg was slightly less than that of monovalent cations. This might be due to the fact that MB exists as MB+ when dissociated, which might show a higher competition with monovalent cations. There was no significant difference between K and Na. The results indicated that cations do not interfere in the MIBK-DCE (3:1) mixed solvent, and, thus, the solvent can be applied to extract AS from a wide range of natural water samples.Anions cause interference when the chloroform method is used, especially when the AS concentration is very low in water samples, because anions in natural water samples can form ion-pairs with the cationic MB(+) dye. This can result in a higher analytical value than the real concentration ,33,37,463−, followed by NO2− and CN−, but those by H2PO4− and HCO3− were relatively small compared to the former three anions. In case of carboxyl group anions, interferences by aromatic compounds were higher than those by aliphatic compounds. In aliphatic compounds, interferences seemed to be higher as numbers of COO− groups increased. Among aromatic compounds, interferences were highest with salicylate, where both COOH and OH groups exist, followed by biphthalate, which has only one COOH group, and benzoate, which has no functional group. Among polyatomic ions, interferences were highest with NOConsidering the concentrations of anions commonly present in natural water samples and tested in this study, anionic interferences on SDS extractability by the MIBK-DCE (3:1) mixed solvent was considered to be minimal, except for the cases of halogen group ions such as I- and Br- that are presumably low in the natural water samples .Criteria for an optimum solvent for the analysis of AS in aquatic samples include the following: a high AS extractability, a low volatility, a density lower than water, a quick separation time for the aqueous and solvent phases, and a low toxicity. The use of any single solvent screened in this study, including chloroform, could not meet these requirements. The combination of a MIBK-DCE (3:1) mixture satisfied such criteria and proved to be an ideal solvent for AS analysis. The extraction efficiency of SDS by MIBK-DCE (3:1) was superior to that of chloroform and was independent of pH, ionic strength, ionic interference, and washing process. The use of a MIBK-DCE (3:1) mixture could be applied for the determination of AS in various water samples, including seawater and industrial wastewater. The MIBK-DCE (3:1) mixture is a suitable alternative compared to traditional standard methods being used today. Development of a new analytical method for AS to replace chloroform, used in the conventional method, should be carried out.