Diabetes is a metabolic disease affecting many tissues and organs. The main etiological factor for diabetic complications is hyperglycemia and subsequent pathologies, such as oxidative stress. One of the organs susceptible to the development of diabetic complications is the eye with all of its elements, including the lens. The aim of this study was to evaluate the effect of silymarin, an extract obtained from milk thistle fruit husks, on the oxidative stress markers in the lenses of type 1 diabetic rats. The study was performed on male rats in which type 1 diabetes was induced with 60 mg/kg streptozotocin injection. Diabetic animals were treated via an intragastric tube with silymarin at 50 and 100 mg/kg doses for four weeks. Multiple oxidative stress and polyol pathway-related parameters were measured in the lenses, and auxiliary biochemical tests in the serum were conducted. Diabetes induced severe pathological changes both in the lenses and the serum, and silymarin counteracted several of them. Nevertheless, the qualitative analyses encompassing all tested parameters indicate that silymarin slightly improved the overall state of diabetic animals. Upon the obtained results, it can be concluded that silymarin reveals a faint positive effect on the lenses in type 1 diabetic rats. Silybum marianum (L.) Gaertn. It is available as dietary supplements and can be registered as a drug [Silymarin is a flavonoid-containing mixture extracted from the seeds and fruits of the milk thistle plant—s a drug ,2,3,4. Ts a drug ,6. Silyms a drug ,8. Aparts a drug , has a ps a drug ,11, actss a drug , and cans a drug , cardioms a drug , or diabs a drug ,15. Clins a drug .Diabetes mellitus is a metabolic disorder characterized by hyperglycemia resulting from insulin deficiency, insulin resistance, or both. Although there are many types of diabetes, two of them are dominant: type 1 and type 2 diabetes . In typeBased on the available literature data, it can be hypothesized that silymarin may also positively affect the lenses in diabetic conditions. Therefore, this study aimed to investigate if the oral administration of silymarin counteracts negative alterations in the lenses of streptozotocin-induced type 1 diabetic rats. The experiment was carried out on the rat model of type 1 diabetes. For the purposes of the study, three-month-old Wistar male rats were purchased from the Centre of Experimental Medicine at the Medical University of Silesia in Katowice. The rats were divided into four experimental groups: NDM—nondiabetic control rats; T1DM—diabetic control rats, in which type 1 diabetes was induced; SIL50—type 1 diabetic rats treated with silymarin at a dose of 50 mg/kg for 28 days; SIL100—type 1 diabetic rats treated with silymarin at a dose of 100 mg/kg for 28 days. Silymarin doses and administration periods were chosen based on the literature data ,31,32. TSilybum marianum fruit husk extract standardized for silymarin content. The drug tablets were pulverized and weighted, then suspended in an adequate amount of water to obtain a 50 or 100 mg/kg dose. The suspension was given by an intragastric tube at a 1 mL/kg volume. The volume of water or silymarin suspension was adjusted according to the current body weight of each animal. All rats were treated for 28 consecutive days. One day after treatment was over, the animals were euthanized by sedation with a single intraperitoneal injection of ketamine and xylazine mixture , followed by the collection of the whole volume of the blood from the heart. The collected blood was then centrifuged in order to obtain the serum for biochemical analyses.Type 1 diabetes was induced in the T1DM, SIL50, and SIL100 groups of rats by a single injection of streptozotocin  at a dose of 60 mg/kg . STZ wasv/w). The obtained homogenate was divided: one part was frozen right after homogenization , while the other was centrifuged for 15 min at 10,000× g and +4 °C. The supernatant was collected from this centrifuged homogenate, while the residue was discarded. Both the supernatant and total homogenate were used for further biochemical analyses.The eyeballs were extracted from the euthanized animals, and then the lenses were separated from the remaining eyeball structures. The lenses were weighted and homogenized manually with a glass homogenizer in a phosphate-buffered saline (PBS), pH 7.4 .Glucose, fructosamine, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, urea BUN, and uric acid levels, as well as the activity of aspartate transaminase (AST) and alanine transaminase (ALT), were measured with the commercially available colorimetric kits . The insulin level was evaluated with an ELISA kit .The protein level in the lenses was assessed using the Lowry’s method  using boSuperoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities were assessed both in the lenses and the serum. For this purpose, commercially available kits were used . Additionally, in the lenses, the activities of glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) were evaluated. GR activity was measured with an appropriate Cayman kit, while G6PD activity was tested with a Pointe Sci. kit . The activity of these enzymes was standardized per protein level: for the lenses, the values of soluble protein were used, as the activity of these enzymes was measured in the supernatant, and the protein level in the serum was measured with BioSystems colorimetric kit.The supernatant collected from centrifuged lens homogenate was also used to evaluate the thiols and glutathione levels in the lenses. Total thiols  level was measured with a method presented by Ellman , and nonThe level of advanced glycation end products (AGEs) in the lenses and the serum was measured using an OxiSelect ELISA kit . The advanced oxidation protein products (AOPP) were measured in the lens supernatant, and the serum using the Witko-Sarsat et al. method  with chlThe supernatant obtained from the centrifugation of the lens homogenate and the serum was used in order to evaluate the following markers describing the oxidative stress: total antioxidant response (TAR), which was assayed according to the protocol presented by Erel , total oThe glucose and fructose levels in the lenses were evaluated by the colorimetric BioSystems kit. Aldose reductase (AR) activity was measured according to the method presented by Patel et al. , and theAll measurements were performed in a Tecan Infinite M200 PRO plate reader with Magellan 7.2 software .p < 0.05. The data in the tables and the figures are presented as arithmetical mean ± standard deviation (SD). The qualitative analysis was performed using a principal component analysis (PCA). PCA was calculated in the Past 3.21 software [https://www.metaboanalyst.ca/ [n = 9, T1DM n = 8, SIL50 n = 8, and SIL100 n = 9. One blood sample from the SIL50 group and one from the SIL100 group hemolyzed, and the serum collection was not possible, thus the number of the samples in serological analyses was n = 7 and n = 8, respectively. This experiment was a part of a large research project, therefore the values for the control groups (NDM and T1DM) are shared with previously published studies [The results obtained from the measurements and adequate calculations were subjected to statistical analyses. As for quantitative analysis, a one-way analysis of variance (ANOVA) followed by a Fisher’s Least Significant Difference (LSD) post hoc test was used. ANOVA and the post hoc were calculated in the Statistica 13.3 software . The results were considered significant if software  using a lyst.ca/  accessed studies ,48,49,50At the beginning of the experiment, on the day of streptozotocin (STZ) injection, the mean weight of rats  in each group was comparable , and theInjection of STZ at a dose of 60 mg/kg to the rats resulted in disruptions of carbohydrate and lipid metabolism . The gluThe aspartate transaminase (AST) activity did not differ among all the analyzed groups, but the activity of alanine transaminase (ALT) was significantly higher in the serum of the T1DM rats than in the serum of the NDM animals. The administration of silymarin to the diabetic animals did not decrease ALT activity in the serum of the SIL50 and SIL100 rats compared to the T1DM rats. This activity remained significantly elevated compared to the NDM rats. No differences in the ALT activity between the SIL50 and SIL100 groups were recorded .The induction of diabetes with streptozotocin resulted in an elevation of total cholesterol and LDL cholesterol fraction (LDL-C) levels with a simultaneous decrease in the HDL cholesterol fraction (HDL-C) level in the serum of the T1DM rats as compared with the NDM rats. The administration of silymarin to the diabetic rats resulted in a reduction of LDL-C and an elevation of HDL-C levels in both the SIL50 and SIL100 rats, but only in the SIL50 rats was the total cholesterol level significantly lower in comparison to the T1DM rats. In the SIL100 rats, the total cholesterol level was not significantly lower than in the T1DM rats, but also it was not significantly higher than in the serum of the NDM or SIL50 rats. There were no significant changes in the triglyceride levels in the serum of all analyzed rats .Both uric acid and urea BUN levels were significantly higher in the serum of the T1DM rats than in the serum of the NDM rats. The administration of silymarin to the diabetic rats did not affect the BUN level when compared with the T1DM animals, thus its level remained elevated in comparison with the NDM rats. The uric acid level in the serum of the SIL50 rats was significantly lower than in the serum of the T1DM and was not different from a statistical point of view from its level recorded in the NDM animals. Even though the level of uric acid in the serum of the SIL100 rats was not significantly lower compared to the T1DM rats, its level did not differ from the uric acid levels observed in the serum of the NDM and SIL50 rats .There were no significant differences in the level of total or soluble proteins in the lenses of all the tested animal groups. What is more, the total protein to soluble protein ratio also was not affected by induction of diabetes or silymarin treatment .The activity of superoxide dismutase (SOD) in the lenses of control diabetic rats (the T1DM group) was significantly higher than in the lenses of the control nondiabetic NDM rats. Treating the diabetic rats with silymarin at doses of 50 and 100 mg/kg resulted in decreased SOD activity in the lenses of the SIL50 and SIL100 rats. As a result, the activity of SOD in the lenses of animals from these groups was not significantly higher than in the lenses of NDM rats. As far as the SOD activity in the serum is concerned, the induction of diabetes in the T1DM animals resulted in a decrease in the activity of this enzyme. Silymarin administration did not counteract the changes in its activity in the serum, thus the serum SOD activity in the SIL50 and SIL100 rats was significantly lower than in the serum of the NDM rats. No differences in the SOD activity in both the lenses and the serum between the two silymarin-treated groups were observed A,D. The In the lenses of the control diabetic rats (the T1DM group), the levels of total thiol groups , protein thiol groups (P–SH), and nonprotein thiol groups (NP–SH) were significantly reduced in comparison with their levels in the lenses of the nondiabetic control NDM animals A–C. MoreIn the serum and the lenses of the type 1 diabetic rats (T1DM), there was an increase in the malondialdehyde (MDA) level as compared to the rats from the control nondiabetic group (NDM). The administration of silymarin at both doses reduced the MDA level, but only in the lenses of the tested animals. The MDA levels in the serum of the SIL50 and SIL100 rats remained unchanged compared to the serum MDA level recorded for the T1DM rats. Thus, this parameter was still elevated when compared with the serum MDA level of the NDM rats. No significant differences in the MDA levels in the serum or the lenses were noted between the two groups receiving different silymarin doses . The oppThere were no significant changes in the total antioxidant response (TAR) in the lenses within all the tested groups. In the serum, TAR in the diabetic control T1DM animals was not different from the TAR value obtained in the serum of the control nondiabetic NDM rats. The administration of silymarin to the rats from the groups SIL50 and SIL100 elevated the serum TAR values compared to their values recorded in the serum of the T1DM animals, and these values did not differ significantly from the TAR serum value observed in the NDM rats A,D. On tAfter diabetes induction, the levels of all sugars from the polyol pathway, i.e., glucose, sorbitol, and fructose, were elevated in the lenses of the T1DM rats when compared to the lenses of the nondiabetic NDM rats. Treatment with silymarin at the doses of 50 and 100 mg/kg did not affect glucose, sorbitol, or fructose levels in the lenses compared to their levels recorded in the lenses of the T1DM rats. Thus, these parameters remained significantly higher than in the lenses of the NDM rats. While in the case of the glucose level in the lenses, there were no differences between the used doses of silymarin; the differences between the SIL50 and SIL100 groups regarding the two other sugars were noted. The sorbitol level in the lenses of SIL50 animals was significantly lower than in the lenses of the SIL100 rats, while the fructose level was lower in the lenses of the SIL100 rats than in the lenses of the SIL50 rats . Aldose As seen in the hierarchical clustering heatmap from the serum parameters A, there A similar situation was observed for the lenses. The hierarchical clustering heatmap prepared for the lenses revealed a distinctively separated cluster formed by the samples originating from the NDM group. As for the other samples—there was an overriding cluster for all diabetic groups—with one secondary cluster formed for several samples from the SIL50 and SIL100 groups (with a predominance of samples from the SIL100 group). As for the second subcluster formed with interwoven samples from the T1DM group and remaining SIL50 and SIL100 rats, there were no other pronounced clusters formed A. As forThe separation of the group clusters was also visible in the PCA plot B. The clThere was also a distinct separation of the SIL50 and SIL100 clusters from the T1DM cluster along the PC2, but as far as the PC1 was concerned, only the cluster formed by the SIL100 was separated from the T1DM cluster in a significant manner and thus was placed in between the T1DM and NDM clusters, but still closer to the T1DM than the NDM cluster. In addition, the SIL100 cluster was significantly separated from the NDM cluster along the PC2, as it was located nearer the bottom part of the plot. It should be highlighted that there were no significant differences between the SIL50 and SIL100 groups with regards to separation along both the PC1 and PC2 . The sepThe lens is a main refractive element of the eye, which enables focusing the light on the retina . It may During diabetes, not only metabolism of carbohydrates is distorted. Diabetic patients also suffer from lipid disorders, which may result in further complications . In the The lens is an insulin-independent organ. In normoglycemic conditions, glucose in the lens is metabolized via glycolysis and, to a lesser extent, by pentose phosphate and polyol pathways ,74. WhenIt is known that persistent hyperglycemia induces oxidative stress, also in the eye . In our Lens macromolecules are susceptible to oxidative damage, which may affect its transparency . Therefo2O2 produced due to SOD activity is reduced. Together with the diminishing GSH amount in the lenses, the level of S-thiolated proteins forming a glutathione-protein heterodimer (PSSG) increases. Proteins can also be S-thiolated by other nonprotein thiols, such as free cysteine or γ-glutamylcysteine forming a protein-S-S-cysteine (PSSC) or protein-S-S-γ-glutamylcysteine (PSSGC) heterodimers, respectively [Interestingly, although in the lenses of diabetic rats the elevated levels of protein oxidation markers were noted, the level of protein, both total and soluble, did not change significantly. Glutathione (GSH) is a crucial nonenzymatic antioxidant within the lens, assuring redox homeostasis . It is aectively . We presSilymarin administration at a dose of 50 mg/kg resulted in a slight increase in the GSH level as compared to the lenses of diabetic, nontreated animals. But when this level was compared to the GSH level noted in the lenses of nondiabetic rats, it was still extremely reduced, as can be clearly seen both in the An enhanced polyol pathway results in the overgeneration of advanced glycation end products (AGEs) in the lenses . In our Additionally, to evaluate the overall effect of silymarin on the serological and lens parameters, we performed statistical analyses encompassing all parameters at one time. Hierarchical clustering heatmaps make it possible to present the data matrix graphically in the form of ordered clusters and help identify groups of correlated samples. Colors used in such heatmaps represent standardized data values. Moreover, because data density is high in cluster heatmaps, it is possible to present large amounts of information compacted into a small space . We obseAs far as the lenses are concerned, the hierarchical clustering heatmap, similar to the serum, revealed a clearly pronounced cluster formed by the NDM group. The rest of the samples were grouped together, and then subclusters were formed. Other information that can be read from this heatmap is that several features assembled in clusters: parameters connected with polyol pathway and levels of all glutathione forms, vitamin C, and nonprotein thiols. These were also the main features deciding the separation along the PC1 visible in the PCA biplot for the lenses. Also, a separation along the PC2 was noted, but different parameters determined it. It should be highlighted that although according to MANOVA, the silymarin doses did not differ between themselves, the administration of the higher dose, 100 mg/kg, resulted in a significant separation of the SIL100 cluster from the T1DM cluster along the PC1. Interestingly, the separation from the T1DM with regard to the PC2 was observed for both silymarin clusters, and the SIL100 cluster also separated significantly from the NDM cluster. The clusters formed by the samples from silymarin-treated groups of animals were located lower in the plot than the T1DM cluster, and the SIL100 even lower than the NDM cluster. We observed a similar separation pattern along the PC2 earlier, examining the effect of two other flavonoids: naringenin and chrysin ,50. In bOur study aimed to investigate the effect of silymarin on diabetes-induced pathological changes in the lenses of rats at the biochemical level. Diabetes, which was induced with streptozotocin, was confirmed by blood glucose level measurements and caused disturbances in oxidative, carbohydrate, and lipid homeostasis in the serum. As expected, silymarin improved the altered parameters connected with lipid metabolism in the serum, but it cannot be regarded as an antihyperglycemic drug in the course of type 1 diabetes. As a result of diabetes, severe oxidative stress and osmotic stress-related pathologies occurred within the eye lens of the rats. Our study revealed that based on the quantitative analyses performed for every single parameter separately, silymarin beneficially affected only several of them. Notably, only parameters connected with oxidative stress were improved, but no effect on the polyol pathway was noted. Nevertheless, when the general picture was evaluated based on the qualitative multivariate analyses, it turned out that silymarin improved the overall condition of the lenses in the diabetic rats, despite the lack of observed quantitative effect in individual parameters. This leads to a conclusion that silymarin affects positively the lenses of diabetic rats at a biochemical level, but the effect is delicate. In order to explain the mechanisms underlying these changes, molecular studies are necessary. Hence, more tests in more complex experimental set-ups are needed, and our research points out the possible direction for future investigations.


FRMR contains three parts: the feeding cell, reacting cell and renewal cell. A flat membrane of polyacrylonitrile (PAN) was used in the reacting cell, which used mixed solutions of kerosene and NaOH. The amine extractant (N1633) was used as the carrier, and simulated mine wastewater containing polytungstate was used as the feeding solution. The influencing factors of the pH and the other ion strengths in the feeding solutions, the volume ratio of kerosene to NaOH solution, and the N1633 concentration in the renewal solutions were investigated in order to obtain the optimum technique parameters. It was found that the removal percentage of polytungstate could reach 92.5% in 205 min, when the concentration of the carrier (N1633) was 0.18 mol/L, the volume ratio of kerosene and NaOH in the renewal cell was 3:2, the pH in the feeding cell was 4.00, and the initial concentration of polytungstate was 3.50 × 10−4 mol/L. The stability and feasibility of FRMR were tested by the investigation of polytungstate retention and the reuse of the membrane.A novel flat renewal membrane reactor (FRMR) with mixed amine extractant N1633 dissolved in kerosene and NaOH solvent was studied for the removal of polytungstate [expressed as W Tungsten is a rare refractory metal with excellent physical, chemical and mechanical properties. Tungsten and its compound products are widely used in the fields of national economy, national defense construction and high-tech industry ,2. They Wastewater containing tungsten and its compounds mainly comes from mining, beneficiation, metallurgy, electroplating, and glass industries ,8. This In past decades, the removal and reuse of heavy metals from wastewater by liquid membranes (LMs) coupled with variations of carriers  were recognized to have broad application prospects, due to their advantages of having a high speed, needing a short time in operation, having a high enrichment ratio, consuming less reagent, and having a low cost ,24,25,26In order to solve the problems of the difficult treatment of polytungstate wastewater and the low efficiency of traditional methods, we proposed a new method based on the traditional supported liquid membrane. At the same time, this new method should overcome the problems of membrane stability and durability. In this work, a novel flat renewal membrane reactor (FRMR) with a mixture of N1633 dissolved in kerosene and NaOH solvent was designed based on SLM to efficiently remove W (VI) from mine wastewater with high stability. N1633 was used as a carrier, and kerosene was used as the organic solvent. The influencing factors on the removal of polytungstate and the stability of the novel FRMR were discussed with the expectation of breakthrough industrial applications in the future. As such, there are still some gaps in ore effluent treatment technology compared to the developed country, while this study will supply the gap with the FRMR in the mine wastewater treatment industry.In brief, the purpose of this study was to solve the problems of polytungstate pollution and its difficult recovery in mine wastewater. This would develop more efficient, more environmental friendly, and more stable polytungstate removal and recovery technology. Simultaneously, it lays a theoretical and scientific foundation for future industrial applications.The framework of the FRMR and its reaction mechanisms are shown in 6W7O24]; (c) the diffusion of the metal complex [([N1633]H)6W7O24] through the membrane from the interface between the membrane and the feeding solution of the feeding part to the interface between the membrane and the renewal solution of the renewal part in the reacting cell; (d) the decomplexation of [([N1633]H)6W7O24] into N1633 and W (VI) in the interface between the membrane and renewal solution, according to chemical Equation (III) in The co-removal involves various (equilibrium) reactions: (a) the diffusion of W (VI) from the feeding part into the interface between the membrane and the feeding solution; (b) the extraction of W (VI) from the feeding solution with the carrier N1633 in kerosene, expressed as chemical Equations (I) and (II) in 3 was used for the regulation of the ion strength in the feeding solution to simulate the industrial wastewater. NaOH was selected as the stripping solution, and the self-made kerosene was used as the organic solvent. Mixed solutions of kerosene with N1633 and NaOH solution were used as the renewal solution. All of the reagents (except the kerosene) were of analytical grade.A flat membrane of porous polyacrylonitrile (PAN) membrane was used in our new design, with a pore size of 0.26 μm, a thickness of 73 μm, a tortuosity of 1.74, and a porosity of 70~80% . Amine extractant (N1633) was used as the carrier in this work, with a density of 0.871 and a purity of 96% . HCl and W (VI) solution was mixed as a feeding solution to simulate the industrial wastewater containing W (VI). HAc-NaAc buffer solution was used for the pH adjustment (2.4–4.2) of the feeding solution, and the mixed solution of NaCl and KNORr) and separation coefficient were calculated as the removal percentage; they were calculated using Equation (1).A Digital Acidity Ion Meter  was used to determine the pH of the solution. The concentration of W (VI) was determined by spectrophotometry with 4-(2-pyridine-azo) resorcinol (PAR) as the chromogenic agent, and the absorbance was measured at 470 nm. The removal efficiency  concentration, at 20, 60, 90, 120, 150, 175, 205 min, respectively.All of the experiments were accomplished at 20 ± 6 °C with the FRMR. The effective area was 40 cm−4 mol/L in the feeding cell. As shown in Basically, the concentration difference between the feed part and the renewal part in the reacting cell is the driving power of the mass removing process, which should be impacted by the pH of the feeding solution . In orde6W7O24], which hypothetically increased the removal efficiency of W (VI) when the pH decreased. Furthermore, when H+ increased, this lead to the formation of the ([N1633]H+Cl−) (Equation (I) in 6W7O24] (Equation (2) in 22− when pH decreased, which hinders the reactions of Equation (II) and lowers the removal efficiency. As a result, the lower the pH is, the more inefficient the removal percentage is. We chose a pH of 4.00 as the optimum pH condition of the feeding part during the following experiments. When the pH was 4.00, the removal percentage of W (VI) was 88.9%.Basically, pH is the driving power of the mass diffusion of the complex [([N1633]H)3 were used to simulate the industrial wastewater, and to investigate the effects of the initial ion strength in the feeding cell on the removal percentage of W (VI). As shown in In the industrial wastewater, there always existed other ions, which may affect the removal of W (VI). Thus, the NaCl and KNOAs we described above, the mixed solutions in the renewal cell were a certain volume ratio of kerosene with N1633 to NaOH. In order to investigate the volume ratio effects of the mixed solutions, the ratios of kerosene to NaOH were adjusted to 1:5, 1:2, 1:1, 3:2, 4:3, 5:2, which is shown in 6W7O24] but also increased the concentration differences of the H+ concentration in the feeding part and renewal part of the reacting cell. Finally, the diffusion of both [([N1633]H)6W7O24] and the recycled N1633 were increased. When the volume ratio of the mixed solutions increased, the complexing rate increased while the stripping rate and diffusion rate decreased. The balance of these reactions resulted in the maximum removal efficiency of W (VI), when the volume ratio of kerosene to NaOH was 3:2. Meanwhile, the final results showed that when the volume ratio was 3:2, the removal percentage of W (VI) was 89.7%.In our work, kerosene with N1633 was selected as the membrane solution, which could recover the stability of the membrane in order to improve the complexing percentage to increase the efficiency of W (VI) removal, by increasing the chances of the recycling of the N1633. NaOH was selected as the stripping solution for the removal of W (VI), the increase of which not only increased the stripping percentage for the decomplexation of [([N1633]H)6W7O24] are. The higher probability of the diffusion and decomplexation of the [([N1633]H)6W7O24] increased the removal efficiency, as shown in Moreover, N1633 played important roles in the removal of the W (VI). Based on Equations (I) and (II) in In the previous investigations of the removal of heavy metal using SLM, the retention of the heavy metal ion on the membrane was observed and concerned ,13. In t6W7O24] in the interface between the membrane and renewal solution with the increase of W (VI) in the renewal solution of the reacting part when the running time increased. With the increase of the running time, the balance reached and the detention of W (VI) no longer increased, with approximately 16% of the W (VI) on the membrane of the reacting cell. However, this did not have negative effects on the renewal of the membrane. As shown in This is due to the decrease of the decomplexation percentage of [([N1633]H)In view of the difficulty of removing polytungstate from mine wastewater, a flat renewal membrane reactor (FRMR) was studied. The running and the influencing factors of this FRMR with a mixture of N1633 dissolved in kerosene and NaOH solvent for the removal of the polytungstate from the simulated wastewater was considered in the laboratory in this work. The results showed that FRMR is able to remove polytungstate from simulated industrial wastewater with high removal efficiency, using the mixture of a carrier of N1633 and NaOH (stripping solution) as a renewal solution. The optimum technology parameters were obtained based on the effects of different influencing factors, with consideration for both the removal efficiency and solvent costs. The resulting optimum parameters of the FRMR were: the concentration of the carrier (N1633) was 0.18 mol/L, the volume ratio of kerosene and NaOH in the renewal cell was 3:2, the pH in the feeding cell was 4.00, and the running time was 205 min. Under the optimum conditions, the removal percentage was 92.5%. Although there is a detainment of polytungstate on the membrane during the operation, the stable retention percentage remained lower than 18%. When the membrane was used six times, the removal rate of polytungstate was still higher than 80%. However, in order to apply the FRMR on a larger scale, the stability and durability of the membrane modules should be solved further in the future.


To evaluate the correlation and clinical significance of T lymphocyte subsets, IL-6 and PCT in the severity of patients with sepsis.One-hundred and twenty patients with sepsis admitted to Baoding No.1 Central Hospital from March 05, 2021 to March 05, 2022 were selected and divided into three groups according to the severity of the disease: the sepsis group, the severe sepsis group and the septic shock group, with 40 cases in each group. The venous blood of all patients was drawn with a sterile vacuum blood collection tube after admission to detect the levels of T lymphocyte subsets CD3+, CD4+, CD8+, CD4+/CD8+, and the venous blood was collected to detect the levels of interleukin-6 (IL-6) and procalcitonin (PCT). The three groups of patients were compared to analyze whether there were differences, and whether there was a correlation between the level of each indicator and the prognosis of patients after treatment.The levels of CD3+, CD4+ and CD4+/CD8+ in the three groups decreased with the aggravation of the disease, with a significant difference (p=0.00). The levels of IL-6 and PCT increased with the aggravation of the disease among the three groups, with statistically significant differences . The better the patients recovered after treatment, the higher the levels of CD4+ and CD4+/CD8+, and the two were positively correlated; While the lower the levels of IL-6 and PCT, the two were negatively correlated.Peripheral blood T lymphocyte subsets and serum IL-6, PCT are abnormally expressed in patients with sepsis, and have a close bearing on the severity of the disease, which has a certain predictive value for patients after recovery. In view of this, the above indicators are of high clinical significance. Sepsis, as a common acute critical disease in the intensive care unit (ICU), is characterized by rapid disease progression, serious consequences and high mortality.A total of 120 patients with sepsis admitted to Baoding No.1 Central Hospital from March 05, 2021 to March 05, 2022 were selected as the study group and divided into three groups according to the severity of the disease: the sepsis group, the severe sepsis group and the septic shock group, with 40 cases in each group. Among them, 17 males and 23 females were grouped into the sepsis group, aged 30-67 years old, with an average of 55.74 ± 9.76 years old. Nineteen males and 21 females were grouped into the severe sepsis group, aged 30-65 years old, with an average of 54.13 ± 11.32 years old. 13 males and 27 females were grouped into the septic shock group, aged 35-70 years old, with an average of 55.89 ± 10.19 years old. There was no significant difference in general data between the three groups, which were comparable . MeanwhiThe study was approved by the Institutional Ethics Committee of Baoding No.1 Central Hospital, and written informed consent was obtained from all participants.All patients who met the diagnostic criteria for sepsis and were confirmed by clinical symptoms, laboratory examination and microbiological examination, etc.Patients under the age of 70;Patients with no previous history of infectious diseases;Patients whose family members are willing and able to cooperate to complete the study;Patients who signed the informed consent.Patients complicated with severe organic or congenital diseases of heart, liver and kidney;Patients whose hospital stay is less than 24 hour and unable to complete the studyPatients with incomplete clinical data;Patients with mental system diseases who cannot cooperate with the completion of the study;Patients who have recently taken relevant drugs such as immunosuppressants that affect the study in the near future.Five ml of venous blood was drawn with a sterile vacuum blood collection tubes for all patients after admission, flow cytometry  was used to detect the levels of T lymphocyte subsets CD3+, CD4+, CD8+, CD4+/CD8+, and venous blood was collected to detect the levels of interleukin-6 (IL-6) and procalcitonin (PCT) . The three groups of patients were compared to analyze whether the levels of CD3+, CD4+, CD8+, CD4+/CD8+, PCT and IL-6 were correlated with the prognosis of patients after treatment. All blood samples were labeled according to the sepsis group, the severe sepsis group and the septic shock group, and were performed by two senior laboratory physicians in strict accordance with the operating procedures. The results were judged by two senior physicians.Observation indicators: 1) The similarities and differences of the levels of T cell subsets, IL-6 and PCT between the study group and the control group were compared and analyzed; 2) The levels of T lymphocyte subsets in the sepsis group, the severe sepsis group and the septic shock group were compared and analyzed; 3) The levels of IL-6 and PCT in the sepsis group, the severe sepsis group and the septic shock group were compared and analyzed; 4) The correlation between different groups in terms of the levels of T cell subsets, IL-6, and PCT after recovery were compared and analyzed. Judgment criteria after recovery: One week after treatment, patients were evaluated according to their symptoms and laboratory examinationχ̄±S). Repeated measurement analysis of variance was used for comparison of test data, LSD-t test was used for pairwise comparison, and 2-test was adopted for rate comparison. Two independent samples t test was used for comparison between groups, and the correlation was expressed by the Pearson correlation coefficient. P<0.05 indicates a statistically significant difference.All the data were statistically analyzed by SPSS 20.0 software, and the measurement data was expressed as , while the levels of PCT and IL-6 were significantly higher than those in the health control group, with a statistically significant difference (p<0.05) .The changes of T lymphocyte subsets among the three groups are shown in The levels of IL-6 and PCT among the three groups increased with the aggravation of the disease, and the difference between the groups was statistically significant  .The level of T lymphocyte subsets was correlated with the recovery of sepsis. The better the patients recovered after treatment, the higher the levels of CD4+ and CD4+/CD8+, and the two were positively correlated, while CD3+ and CD8+ have no obvious correlation with the recovery of sepsis . The levSepsis, as a common serious infectious disease, has become a common cause of death in critically ill patients. its pathogenesis is the imbalance of the dynamic balance between inflammation and pro-inflammatory reaction, which leads to immune dysfunction of the body, and then induces the body to produce inflammatory factors and inflammatory mediators, which may eventually lead to insufficient tissue perfusion, organ dysfunction, etc.According to animal experimental studiesPCT is secreted by thyroid C cells. In healthy people, the level of serum PCT is considerably low. In contrast, in local infection, viral infection and autoimmune diseases, the level of PCT is slightly increased or not increased, and PCT is released in large quantities under the promotion of inflammatory factors in the body when the body is seriously infected with bacteria or sepsis. Moreover, the level of PCT increased further with the aggravation of the disease, and decreased with the improvement of the disease. It can therefore be seen that PCT has good distinguishing characteristics to distinguish bacterial inflammation from viral inflammation and obtain results quickly.20It was concluded by Ma et al.It was suggested in our study that the levels of PCT and IL-6 were significantly higher than those in the health control group (p<0.05). The levels of IL-6 and PCT increased with the aggravation of the disease . The levels of PCT and IL-6 were correlated with the recovery of sepsis, that is, the lower the levels of PCT and IL-6, the better the patients recovered after treatment, showing a negative correlation.It includes fewer patients were included in the study, and only the indicators of patients with different severity after admission are compared and analyzed, without more systematic post-treatment review and follow-up. In addition, the guiding significance of each indicator for the treatment and the judgment of the effect are not involved in this study. In view of this, proactive countermeasures are being taken to further improve the data, increase the number of samples and dynamically review the changes of various indicators after treatment, so that the study can be further enriched, and the follow-up can be increased, hereby demonstrating the guiding value of various indicators involved in the study for treatmentPeripheral blood T lymphocyte subsets and serum IL-6, PCT are abnormally expressed in patients with sepsis, and have a close bearing on the severity of the disease. Some indicators can have certain predictive value for the recovery of patients. In view of this, the above indicators are of high clinical significance.QL &WY: Designed this study and prepared this manuscript,and are responsible and accountable for the accuracy or integrity of the work.SL: Collected and analyzed clinical data.HL: Data analysis, significantly revised this manuscript.


The literature has described comorbidities among the symptoms of children with Attention Deficit and Hyperactivity Disorder (ADHD) and the auditory processing changes, and these symptoms have been overlooked in the assessment, and consequently, on the rehabilitation of these individuals.To compare the findings of the long latency auditory evoked potentials in children with and without ADHD.This is a historical cohort cross-sectional case-control study, in which we enrolled 30 children with and without ADHD, aged 8-12 years. We performed the long-latency auditory evoked potential test in two scanning procedures through passive and active tasks differing in frequency and duration (MMNf and MMNd) (P300f and P300D).When comparing the performance of children with and without ADHD in the electrophysiological test assessment of hearing, we found significant differences concerning the P2 amplitude in the LE - which was higher for the ADHD group; and concerning the N2 amplitude and latency - which were abnormal in the ADHD group.This study provided a greater understanding of the central auditory pathways of children with and without ADHD when evaluated from electrophysiological tests. Neuropsychological studies have reported that individuals with attention deficit and hyperactivity disorder (ADHD) have alterations in their prefrontal cortex and in subcortical structures, associated to frequent levels of inattention, impulsivity, hyperactivity, disorganization and social awkwardness, involving an inhibitory system deficit or in the working memory executive functionsThe literature has described comorbidities between symptoms of children with ADHD and auditory processing (AP) disorders, and such symptoms have been overlooked in the assessment and consequently the rehabilitation of these individualsAuditory processing disorder is common in children with ADHD and may be due to a deficit in the functioning of the auditory pathway, caused by changes in some of the central auditory nervous system (CANS) structures, or in the cerebral hemispheres, which can be observed by Long Latency Auditory Evoked Potentials (LLAEP), which assesses the auditory pathway integrity from the periphery all the way to the auditory cortexAccording to the DSM-IVMany studies have suggested that attention dysfunction is not the main cause of behavioral changes in individuals with ADHDGiven the above, this study aimed to compare the findings of the auditory evoked potential latencies in children with and without Attention Deficit and Hyperactivity Disorder (ADHD).This study project was submitted to the analysis and appreciation of the Ethics in Research Committee of this University and carried out after approval according to protocol number: 0094/2011. This historical cohort study was cross-sectional and of the case-control type.•Control Group (CG) - comprising 15 children with good academic performance, selected by the teachers following the criterion of satisfactory performance on two consecutive marking periods in a reading and writing assessment;•Study Group (SG) - composed of 15 children properly diagnosed with ADHD by a multidisciplinary team, which included speech and hearing, neurological, educational, neuropsychological and educational assessments, considering the presence of at least six (or more) symptoms of inattention and six (or more) symptoms of hyperactivity-impulsivity persisting for at least six months, according to the Diagnostic Criteria for Attention Deficit/Hyperactivity Disorder from the DSM-IV. We employed Instruments from the neuropsychological battery of tests: WISC-IIIThe study included 30 children of both genders aged 8-12 years, divided into:The children from both groups were evaluated after their guardians signed the Consent Form. All the children had chronological age between 8 and 12 years and were previously submitted to audiological, ophthalmological and psychological evaluations. Thus, we excluded from the study those individuals who did not have audiometric thresholds within the normal range (20 dB HL)& KaplanBasic audiological evaluation was performed in a soundproof booth. For pure tone audiometry we used the GSI 61 (ANSI 3.6-1989 and S3.43-1992 standards) audiometer with TDH-50 phones. Hearing thresholds were obtained by air conduction, in the sound frequencies of 250-8,000 Hz. The normality criterion used was the classification proposed by Lloyd Evaluation of the Long Latency Auditory Evoked Potential was performed using the Biologic Navigator Pro and recorded with five disposable electrodes positioned at Fz and Cz in reference to the right (A2) and left (A1) lobes, using the two recording channels of the equipment, the ground electrode was placed on Fpz. Impedance was maintained at a level below 5 KW.The components were surveyed in two sweeps, i.e., it was first elicited for tonal stimuli (tone burst) differing in frequency - MMNf and P300f , and later, for stimuli differing in duration - MMNd and P300D .Both stimuli differing in frequency and duration were randomly presented in an oddball paradigm, a rate of 1.1 stimuli per second, with a rare stimulus occurrence probability of 20% of the total 250 stimuli. The analysis time of the waves was 500 ms with a filter from 0.5 to 30 Hz and 50,000 mV sensitivity and alternating polarity.For the MMN recording, the patient performed a passive task and was instructed to remain seated and relaxed but awake and watching a video (without sound) to get distracted and not pay attention to the sound stimulus presented to him. As for the P300 recording, the patient should undertake an active task, paying attention and discriminating the stimuli naming them as “thin” during P300f and “short” in P300d.The stimulus presentation was randomized concerning the stimulated ear, alternating them to avoid result biases. Moreover, due to the difficulties inherent to the behavior of children with ADHD, we decided to replicate the test only when necessary, in routine use the Cz and Fz records, in order to verify and ensure data accuracy.For final result analysis, we chose to use the records obtained at Cz, since in this study this was the region where the records were better; in addition, the literature has consistently described it as the region with the best visualization of these potentials.& Colafêmina•Identification of the N1-P2-N2 complex - the first three waves that appear in the sequence and have negative - positive - negative polarity, respectively, occurring in the replication of the traces, frequent and rare, between 60 and 300 ms;•P3 identification - the largest positive wave - immediately after the N1-P2-N2 complex, occurring in tracing replication for rare stimulus, between 240 and 700 ms;•Latencies were marked on the highest peak, i.e. the point of maximum wave amplitude;•The amplitudes were marked from the peak of the wave to the base line, and in the case of the N2-P3 inter-amplitude;In order to identify the P300 wave, we used the criterion proposed by Junqueira To identify the MMN waves, we considered the biggest wave of negative polarity, between latency values from 100-300 ms, viewed by the subtraction of the rare stimulus tracing from that of the frequent stimulus tracingp-value, and the level of significance adopted was always 5% or 0.05 (p ≤ .05).The result was described as Upon assessing the electrophysiological evaluation of hearing during the active task with stimuli that varied on frequency, P300f, the right ear (RE) had statistically significant difference only in the N1 latency, while in the left ear (LE) there was no difference as to the level of significance in any of the variables.p-value of the N1, P2, N2, P3 amplitude and latency values; and N2-P3 inter-amplitude in the P300f assessment in both groups of RE and LE, respectively.In the P300 evaluation, when we stimulated with a stimulus that varied in duration, P300d, the RE had no statistically significant difference when the two groups were compared with and without ADHD; while for the LE it was significant when comparing the P2 and N2 amplitude and N2 latency.p-value of the N1, P2, N2 and P3 latency and amplitude variables; and P300d N2-P3 inter-amplitude for the RE and LE, respectively.Comparing the electrophysiological evaluation of passive listening with stimulation with varied frequency, MMNf, both ears showed no difference as to the level of significance when the two groups - with and without ADHD were compared.In assessing the MMN stimulation with varying stimulus for duration, MMNd, we did not find a statistically significant difference when comparing the two groups with and without ADHD in any of the variables in both ears.There are many studies that have evaluated the P300 in children with ADHD, but few have focused on the other LLAEP components: N1, P2 and N2Concerning the P300d assessment, the LE had better P2 amplitude for the SG when compared to the CG, which corroborates studiesThe higher P2 amplitude in the SG children can be justified by studiesIn evaluating the P300d for the LE, we found a significant difference for the N2 amplitude, in which the CG had higher negativity when compared to the SG, and in N2 latency - where the SG values were more elongated. These results corroborate other studiesWith regard to N2 latency, the LE also showed a statistically significant difference when the SG was compared to the CG, and we found longer latency values for the SG, corroborating other studiesIn this study, we observed that the N2 was the only component to show significant differences in terms of both latency and amplitude when children with ADHD were compared to those without it, which makes us consider that the ADHD children of our study had a decline in the efficiency of responses involving pre-attentional and discriminatory processesConcerning the P3 component findings, it is consistent with studiesA first explanation for not having significant differences between the P300 in children with and without ADHD in this study is that our sample was small, and this is a limiting factor vis-à-vis the specific discussion of these findings. Such a limitation in this study was described by BraynerAnother possible explanation is that children with ADHD have alterations in their pre-attentional and discriminatory processes - which was evident in the results found in the N2 wave; however, these children began to process this information in some way, which could be aided by other structures of the central nervous system and/or by interference of neural plasticity, in which sensory experiences bring about a better neural synchrony or a reorganization of the nerve cellsWith regard to the MMN, both the MMNf and the MMNd did not show statistical difference for any of the variables, either in amplitude or latency when comparing the two groups. This suggests that children with ADHD in our study have deficits when they need to performa a discrimination task, while keeping sustained attention, or maintain attention for an extended period of time. To obtain the MMN, we need pre-attentional processes that are independent of the subject's response; thus, these children did not need to perform any task, which contributed to the normal MMN results found in the present studyAccording to the specialized literatureIn this study, we also found that the larger number of altered results came from the LE stimulation, leading us to suggest that, just like the processing of nonverbal stimuli, and the stimuli varying according to duration, are first processed by the right hemisphereIn comparing the performance of children with and without ADHD in the electrophysiological assessment of hearing, there were significant differences vis-à-vis the P2 amplitude in the LE, which was higher for the group with ADHD and for N2 amplitude and latency of the LE, which were abnormal in the ADHD group.Regarding P300 and MMN, there were no significant differences when comparing both groups.This study provided a greater understanding of the central auditory pathways of children with and without ADHD when evaluated with electrophysiological tests; however, further studies are needed, especially in the national literature, to better understand the functioning of the auditory pathway of these populations.


The aim of this study was to determine the relationship between the altitude of residence and the low birth weight (LBW) of the children of pregnant Peruvian women using a nationally representative database. An analysis of individual-level data from the last 13 years (from 2009 to 2021) of the Demographic and Family Health Survey was performed. The outcome variable was LBW, defined as birth weight less than 2500 g, while the independent variable was the altitude of residence in meters above sea level (masl). To estimate the association between the two variables, the crude and adjusted generalized linear model of the Poisson family with a log link was used along with crude and adjusted prevalence ratios, which were estimated with their respective 95% confidence interval. A total of 151,873 women aged 15–49 years were included between 2009 and 2021. The pooled proportion of LBW was 7.0%. As the main finding, the children of mothers residing at an altitude from 2500 to 3499 masl and ≥3500 masl had a higher probability of LBW. It was found that the children of mothers residing at an altitude above 2500 masl were more likely to have LBW. Our results will help to strengthen the cultural practice of maternal health care and increase its coverage in women residing in high-altitude regions. According to the World Health Organization (WHO), low birth weight (LBW) is a public health indicator that encompasses maternal health and nutritional problems, deficiencies in health care during pregnancy, and socioeconomic conditions . LBW is The biomedical literature suggests that factors such as fetal sex and maternal and socioeconomic characteristics can influence the presentation of LBW in a newborn ,11,12,13In Peru, high-altitude regions (Andes region) are characterized by their location in rural areas and at an altitude exceeding 2500 masl ,27,28,29http://iinei.inei.gob.pe/microdatos/, accessed on 20 November 2022). The 2009 ENDES was used because the survey has been conducted annually since that year. Briefly, the ENDES is a nationally representative survey that uses a complex sampling design and collects information on sociodemographic and health indicators of women of childbearing age and their children under 5 years of age. The sampling methodology used in the ENDES is provided in detail in the technical reports of the survey [The present study used individual-level data from the Demographic and Family Health Survey (ENDES—acronym in Spanish) conducted between 2009 and 2021  ,44,45,46Women selected for the individual questionnaire for women of childbearing age were included in this study. The individual questionnaire collected information on demographic and social characteristics, reproductive history, contraceptive use, maternal and child health, fertility, and domestic violence, among the main topics ,44,45,46The study sample included women of childbearing age between 15 and 49 years who had had a child within the last five years at the time of the survey. Likewise, women with a residence time of 5 years or more were included. Women who did not reside in the altitude cluster at the time of pregnancy were excluded.The outcome variable of this study was LBW coded as “1” if the mother reported that the child was born weighing less than 2500 g and “0” otherwise. This classification was based on the definition given by the WHO [The exposure variable was the altitude of the cluster where the dwelling was located. In the ENDES, specifically since 2015, the measurement of the altitude was given in masl using an application to obtain georeferenced data  through a global positioning system measured at one meter from the front door of the respondent’s home ,50,51,52Covariates were selected based on previous studies and data available in the ENDES ,33,54. TA combined database of the 13 years (from 2009 to 2021) of the ENDES was constructed and used for analysis. We used Stata version 17.0 (StataCorp) for data management and analysis. Prior to any statistical analysis, the data were weighed using the sampling weight, PSU, and strata to consider the representativeness and sampling design of the ENDES by using the ‘svy’ command. To specify the sample design, a strata and cluster identifier was created for each year of the ENDES, and the sample weight was divided by 1,000,000.p-value of <0.05 was considered statistically significant.Initially, a descriptive analysis was performed to report the characteristics of the participants, including the frequency of LBW, the frequency of women by their altitude of residence, and the covariates of interest using absolute and relative frequencies for each year which were pooled. Next, the crude and adjusted generalized linear model of the Poisson family with a log link was used to evaluate the association between LBW and the altitude of residence. The association results are expressed as prevalence ratios (PR) and their 95% confidence intervals (CI). The possible problem of multicollinearity was evaluated by performing a variance inflation factor test which found no correlation between the independent variable and covariates. A http://iinei.inei.gob.pe/microdatos/ (accessed on 20 November 2022) were used. The process of downloading and using the ENDES databases is described in the study by Hernández-Vásquez and Chacón-Torrico [Approval from a research ethics committee was not sought for this retrospective study because only anonymized secondary data that are in the public domain and available at -Torrico . ENDES pA total of 151,873 women aged 15–49 years were included between 2009 and 2021. Of the total participants included, 51.3% of the children were male, and 47.6% of the mothers belonged to the 25–34 age group. In addition, 26.3% of the mothers were nulliparous, and most of the mothers belonged to the middle (22.1%) and poor (21.8%) wealth quintiles and resided in an urban area (76.4%). On the other hand, the highest proportions of LBW were observed in the years 2010 (8.0%), 2013 (7.9%), and 2015 (7.5%), while the lowest proportions were found in the years 2014 (6.2%), 2019 (6.2%), and 2021 (6.5%). Further details of the characteristics of the population included according to the year of the survey are shown in Overall, the pooled proportion of LBW between 2009 and 2021 was 7.0%. Likewise, the highest prevalence of LBW was observed in female children (7.4%) whose mothers belonged to the 15–24 age group (7.8%) and who were their first child (7.6%). Additionally, mothers residing between 3500 and 3499 masl (9.0%), belonging to the poorest wealth quintile (9.3%), and in a rural area (8.5%) had the highest proportion of children with LBW .On average, we found that 71.6% of the mothers resided at an altitude lower than 1500 masl, followed by 14.0%, 7.9%, and 6.4% of the mothers residing at an altitude from 2500 to 3499 masl, ≥3500 masl, and from 1500 to 2499 masl; respectively . RegardiIn the crude model, the LBW was associated with an altitude of residence from 2500 to 3499 masl and ≥3500 masl. Likewise, the same pattern was found in both the adjusted and the crude models. Specifically, model 1, which was adjusted for the mother’s age, child’s sex, wealth quintile, nulliparity, and area of residence, found that the children of mothers residing at an altitude between 2500 and 3499 masl  and ≥3500 masl  had a higher probability of having LBW, while in model 2, when adjusted for all variables in model 1 and the survey year, the same pattern was identified, with the children of mothers residing at residence altitudes of 2500–3499 masl  and ≥3500 masl  demonstrating a higher probability of presenting LBW .The sample weights and sample design of ENDES were included for all the estimates.Model 1: Model with the age group of the mother, sex of the child, nulliparity, wealth index, and area of residence.Model 2: Model with the age group of the mother, sex of the child, nulliparity, wealth index, area of residence, and year of the survey.Our study sought to determine the association between the altitude of residence in Peruvian mothers and the presence of LBW in their children by performing an analysis comprising 13 years of a nationally representative survey. We found the pooled proportion of LBW to be 7% in the study period. As the main finding, we observed that after adjusting for potential confounders, mothers residing at an altitude of 2500–3499 masl and ≥3500 masl had a higher probability of having a child with LBW compared to those residing at an altitude lower than 2500 masl. It should be noted that the temporality of this association was based on the assessment of LBW in the last child under five years of age.We found that 7% of newborns were found to have LBW in the study period, with LBW ranging from 6.2% in 2014 and 2019 to 8.0% in 2010. This finding is lower than that reported in studies conducted in high-altitude regions of the United States (11.8%)  and NepaThe identified association between the altitude of maternal residence and LBW has previously been reported in the biomedical literature ,23,33,54Our results could have an impact on clinical practice and public health. In clinical practice, the association between the altitude of residence and LBW observed in our study could allow health personnel to better follow and comply with prenatal check-ups of pregnant Peruvian women residing at a high altitude (>2500 masl). On the other hand, maternal health care that seeks to reduce negative perinatal outcomes in rural or high-altitude regions should implement an intercultural approach since native women (Aymara or Quechua) have ancestral practices that generate a negative perception of maternal health care, resulting in a higher frequency of home births attended by midwives and lower use of formal health services . In addiOur study has limitations and strengths. First, one of the main limitations is the cross-sectional design of our study, which prevents establishing causality between the variables of interest due to a lack of temporality in their measurement. Therefore, longitudinal studies are required to determine the causality between the exposure variable  and the outcome variable (LBW). Second, as birth weight is a variable self-reported by the mother, there could be a recall bias or inaccuracy in the data since the interviewer requests the exact amount of the newborn’s weight from the last pregnancy in the last five years. Third, by using different years of the ENDES, different sample frames were obtained that could have generated differences in the characteristics of the mother and child between each year, and this may have been more notable in the 2020 and 2021 ENDES, which collected part of the information by telephone due to the COVID-19 pandemic ,46. FourIn conclusion, it was found that mothers residing at an altitude above 2500 masl were more likely to have children with LBW. In this sense, the findings of this study will allow the development of strategies that include the cultural practices of women and ensure greater coverage and control of pregnant women to reduce the prevalence and consequences of LBW throughout the life of the newborn.


A number of genomic variants related to native American ancestry may be associated with an increased risk of developing Acute Lymphoblastic Leukemia (ALL), which means that Latin American and hispanic populations from the New World may be relatively susceptible to this disease. However, there has not yet been any comprehensive investigation of the variants associated with susceptibility to ALL in traditional Amerindian populations from Brazilian Amazonia. We investigated the exomes of the 18 principal genes associated with susceptibility to ALL in samples of 64 Amerindians from this region, including cancer-free individuals and patients with ALL. We compared the findings with the data on populations representing five continents available in the 1000 Genomes database. The variation in the allele frequencies found between the different groups was evaluated using Fisher’s exact test. The analyses of the exomes of the Brazilian Amerindians identified 125 variants, seven of which were new. The comparison of the allele frequencies between the two Amerindian groups analyzed in the present study  identified six variants  that may be associated with susceptibility to ALL. These findings contribute to the identification of genetic variants that represent a potential risk for ALL in Amazonian Amerindian populations and might favor precision oncology measures.  ARID5B, IKZF1, PIP4K2A, CEBPE, GATA3, BMI, and CDKN2A genes [Acute Lymphoblastic Leukemia (ALL) is the most common cancer in children and is the principal cause of childhood mortality due to malignant disease ,2. The g2A genes ,9,10,11.Most GWASs addressing ALL susceptibility have focused on homogeneous populations in regions such as Europe or North America, whereas the highest incidence of childhood ALL is found in populations with a major component of native American ancestry, such as the Latin American and hispanic populations of the New World ,12,13,14Given this, in the present study, we investigated the genetic variants potentially involved in the etiology of ALL in traditional Amerindian populations from Brazilian Amazonia. For this, we used New Generation Sequencing (NGS) to define the exomes of 17 genes associated with susceptibility to ALL in samples obtained from indigenous groups that inhabit Brazilian Amazonia, in both ALL patients and cancer-free individuals. The variants encountered in this initial analysis were compared with the data available on populations representing five different continents, which were obtained from the 1000 Genomes database.The present study was approved by Brazilian National Committee on Research Ethics—CONEP (identified by No 1062/2006 and 123/1998). All participants signed a free-informed consent as well as the tribe leaders and when necessary, a translator explained the project and the importance of the research. Their materials were collected according to the Declaration of Helsinki.The study sample included 59 cancer-free Amerindians and five individuals diagnosed with ALL. All the participants were members of isolated ethnic groups located in Brazilian Amazonia . The genhttps://www.1000genomes.org, accessed on: 20 February 2021) to provide comparisons with ethnic groups from other continents. This sample included 661 individuals from Africa (AFR), 503 from Europe (EUR), 347 from the Americas (AMR), 504 from East Asia (EAS), and 489 from South Asia (SAS).Data were obtained from the 1000 Genomes Project database  using the NextSeq 500 high-output v2 300 cycle kit . Samples of 5 mL of peripheral blood were collected from each of the participants of the study. The genetic material was extracted from these blood samples using the Roche Applied Science DNA extraction kit  following the manufacturer’s instructions, and it was quantified using a NanoDrop 1000 spectrophotometer . The exome libraries were prepared using the commercial Nextera Rapid Capture Exome kit  and the SureSelect Human All Exon V6 kit , with the manufacturer’s protocol being followed in both cases. The sequencing reactions were run in the NextSeq 500®, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil) software was used to analyze the annotations of the variants. The variants were annotated in three databases—SnpEff v.4.3.T, Ensembl Variant Effect Predictor (Ensembl version 99), and ClinVar (v.2018–10). The SIFT (v.6.2.1), PolyPhen-2 (v.2.2), LRT , Mutation Assessor (v.3.0), Mutation Taster (v.2.0), FATHMM (v.2.3), PROVEAN (v.1.1.3), MetaSVM (v.1.0), M-CAP (v.1.4), and FATHMM-MKL databases were used for the in silico prediction of pathogenicity.The bioinformatic analyses followed the approach described by Ribeiro-Dos-Santos et al.  and Rodrp ≤ 0.05 significance level was considered for all the analyses.For the statistical analyses, the cancer-free Amerindians were assigned to the Native (NAT) group, while the ALL patients were in the ALL_NAT (Native with ALL) group. All the analyses were run in the R v.3.5.1 program. The differences in the allelic frequencies between the NAT and ALL_NAT were evaluated using Fisher’s exact test, which was also applied to the comparisons with the continental populations. A The analysis of the exomes of the 64 Amerindians investigated in the present study revealed the presence of 125 variants, seven of which were new. The SnpEff software  was usedAs some of the variants were not covered adequately in one or other of the groups (NAT or ALL_NAT), they were excluded from the comparison of allele frequencies. This left 64 variants that were included in the analysis of association. The frequencies of these variants are compared between groups in PIP4K2A (variants rs2765997 and rs1053454), CDKN2A (rs11515), IGF2BP1 (rs8068981), USP7 (rs2304465), and WWOX (rs3764342). The allele frequencies of these six variants recorded in the ALL_NAT group were also compared with those recorded in the 1000 Genomes Project for the five continental populations . The comparisons are shown in p values, in The analyses revealed significant differences between the two groups in six variants of five genes: CDKN2A gene. The frequency of the rs8068981 variant of the IGF2BP1 gene was also significantly different from that of the EAS population, while the frequency of the rs2765997 variant of the PIP4K2A gene was significantly different from those of the AFR and EAS populations.The analyses revealed significant differences between the Amerindian group (ALL_NAT) and all the continental populations  in the frequency of the rs11515 variant of the The incidence of ALL is relatively high in populations with a high degree of native American ancestry, such as Latin American and Hispanic populations, which has been attributed, in part, to the contribution of molecular markers associated with a high risk of ALL in these populations ,13,14,21IKZF1, IKZF3, WWOX, and ZPBP2), most of which were present in the ALL_NAT group, that is, Amerindian ALL patients. These genes are known to have alleles associated with some level of risk in the etiology of ALL in different populations [During the study, we identified seven new variants in four genes  given the significant differences in the frequencies recorded in the two Amerindian groups , and between the ALL group and populations of other groups from around the world.We also identified six variants of five genes that we associated with susceptibility to ALL (CDKN2A gene plays an important role in leukemogenesis. The rs11515 variant is located in the 3′-untranslated region (UTR) of the CDKN2A gene, and is known to be associated with a number of different types of cancer [The f cancer , includif cancer , glioblaf cancer , melanomf cancer , and colf cancer . HoweverPIP4K2A gene have identified an association with susceptibility to ALL [PIP4K2A gene, given that both were more frequent in the Amerindian population, in comparison with the other continental populations, which may reflect a potential correlation with the risk of ALL. A number of previous studies of genetic polymorphisms in the y to ALL ,31. We iIGF2BP1 protein is expressed in a number of different types of cancer, including leukemia. In a recent study based on in vivo and in vitro analyses, Elcheva et al. [IGF2BP1 and the aggressiveness of leukemia, through the persistence of tumorigenicity by increasing critical transcriptional and metabolic regulators. These authors also emphasized that the IGF2BP1 gene is often positively regulated in many types of malignant disease, and is not expressed in most normal tissue, which means that it is a potentially important target for anticancer therapy. The results of the present study indicate that the rs8068981 variant of the IGF2BP1 gene is present at similar frequencies in the Amerindians with ALL and the European population, but contrasts with the Asian populations.The a et al.  found evWWOX gene, which has a suppressor role in solid cancers, with the loss of function resulting in alterations in the adhesion of the cancerous cells to the extra-cellular matrix, which affects cell migration and metastasis. A number of studies have contributed to the description of the role of this gene in leukemic malignancies. Luo et al. [WWOX mRNA and protein is significantly reduced or absent in cases of leukemia and their cell lines in comparison with the controls, which is consistent with the findings of Cui et al. [We also identified in the Amerindian population a positive association with the rs3764342 variant of the o et al.  found thUSP7 gene was also more frequent in Amerindian patients. Jin et al. [USP7 and a lack of ubiquitination and the stabilization of the NOTCH1 oncogene, which contributes to the control of the cell growth of the T cells in lymphoblastic leukemia. In the present study, the rs2304465 variant of the n et al.  found anThe present study is the first to investigate the exome of genes involved in the etiology of Acute Lymphoblastic Leukemia (ALL) in Amerindian populations from the Amazon region. The results of the study provide important genetic data related to the etiology of ALL in such population in which genetic investigations are scarce. The study also contributes to the identification of variants of potential risk involved in the etiology of ALL in Amerindian populations, as well as in other Brazilian populations formed through a high level of admixture with this indigenous group.


O2 uptake from the atmosphere accounts for virtually all the O2 consumption in the avascular cornea; however, a detailed spatiotemporal profile of corneal O2 uptake (COU) remains undetermined. Here, we used a non-invasive self-referencing optical fiber O2 sensor—the scanning micro-optrode technique (SMOT)—to report the O2 partial pressure and flux variations at the ocular surface of rodents and non-human primates. In vivo spatial mapping in mice revealed a distinct COU, characterized by a centripetal gradient with a significantly higher O2 influx at the limbus and conjunctiva regions than at the center of the cornea. This regional COU profile was reproduced ex vivo in freshly enucleated eyes. The centripetal gradient was conserved across the following species analyzed: mice, rats, and rhesus monkeys. In vivo temporal mapping in mice showed a significant increase in the O2 flux in the limbus in the evening compared to other times. Altogether, the data unveiled a conserved centripetal COU profile, which may be associated with the limbal epithelial stem cells residing at the intersection of the limbus and conjunctiva. These physiological observations will serve as a useful baseline for comparative studies with contact lens wear, ocular disease, diabetes, etc. Moreover, the sensor may be applied to understand the responses of the cornea and other tissues to various insults, drugs, or changes in the environment.Oxygen (O The reference value was subtracted.The [2 concentration spatially, i.e., ~20–50 μm, and temporally, i.e., ~2 s, and for the measurement of O2 fluxes, the resolutions achieved were ~60–100 μm spatially and ~11–22 s temporally, respectively.The SMOT system thus achieved a high resolution for the measurements of OThe anesthetized mice with hydrophobic wells around the eye (in vivo) or enucleated eyes in wells (ex vivo) were brought to the measurement stage. A calibrated probe was carefully moved into the measuring medium under the camera and, using a 3D micro-positioner, the micro-optrode was manually moved into the measuring buffer solution in the same view of the region of measurement. The motion controller and ASET software were used to finely move the tip of the optrode to the same focal plane as the region of measurement at about 10 μm perpendicular from the surface. The moving step of the optrode was set to 30 μm between the near and far measuring points, as shown in 2 uptake. Measurements were taken on the cornea  and sclera (conjunctiva), as shown in Three male C57BL/6 mice were used to measure the circadian rhythm of corneal Op-value < 0.05. All data analysis, graphs, and statistical calculations were conducted using Microsoft Office Excel.Data are expressed as mean ± standard error of the mean (S.E.M.). Differences between groups were compared using the parametric Student t-test, and statistical significance was considered if the pO2 levels with a micro-optrode. After anesthesia of the mice, we positioned the probe in the center, periphery, and limbus of the cornea and in the conjunctiva of the sclera, as shown in pO2 of readings were ~20%, which indicated the dissolved O2 saturation in the measuring BSS+ buffer, as shown in pO2 values dropped significantly, as shown in pO2 levels decreased to ~75% of the saturation level at the center and periphery of the cornea, ~65% at the limbus, and ~55% conjunctiva, all significantly lower than the pO2 level at the reference position (>1 mm away from the ocular surface), as shown in pO2 values during the excursion of the probe , as shown in 2 flux following Fick’s first law of diffusion, as shown in 2, following a centripetal gradient of O2 uptake. The uptake of O2 of the limbus and conjunctiva was substantially higher than that of the center and periphery of the cornea, as shown in 2 influx, i.e., the corneal O2 uptake. The calculated COU showed a striking centripetal gradient, with the highest uptake being at the outer edges (limbus and conjunctiva) and the lowest being at the center of the ocular surface, as shown in To seek the putative regional differences in COU across the ocular surface of in vivo mice, we mapped the w/v). This treatment resulted in virtually no detectable COU, as shown in Next, we asked whether the spatial profile of the COU in vivo could be reproduced ex vivo in the cornea, since ex vivo corneal cultures and mouse eyeball cultures are used frequently in the research of ocular surface biology. This reproduction is important as it will expedite experiments with large animals and when in vivo measurements are unfeasible or impractical. We observed a consistent centripetal gradient in the COU ex vivo, with significantly higher uptake in the limbus and conjunctiva than at the center and periphery of the cornea B. This s2 uptake in the different species analyzed, as shown in 2 uptakes in the rat and rhesus monkeys than in the mice, which seemed to roughly correlate with the size of the eyeball, as shown in To gain insights into the evolutionary conservation of the spatial centripetal gradient across the ocular surface, we started by measuring the COU in another rodent, i.e., rats. The ex vivo measurements revealed the same spatial profile with a significantly larger COU at the limbus and conjunctiva when compared to the center and periphery of the cornea, as shown in Therefore, the COU profile was conserved across the rodents and the non-human primates, suggesting an evolutionary conservation that may apply to other mammals.2 uptake in the in vivo mouse eyes in the morning (9 am), at midday (1 pm), and in the evening (5 pm). We observed a similar significant centripetal COU gradient in the three temporal periods, as shown in 2 uptake shifted significantly in the evening, with a ~20% increase in the COU when compared to the morning and midday levels with statistical significance, as shown in 2 uptake at the periphery appeared to increase in the evening, as shown in To test whether the COU varies temporally during the time of day, we measured the O2 uptake at the ocular surface of in vivo mice and ex vivo mice, rats, and rhesus monkeys. We generated a spatiotemporal COU profile at the ocular surface, which was conserved across the mammalian species evaluated (rodents and non-human primates). Spatially, we found a centripetal gradient of O2 influx, with the highest uptake at the limbus and conjunctiva, which decreased towards the center of the cornea. The O2 uptake was absent in the fixed tissues and decreased in the eyes kept at low temperatures, which confirmed that the measurements were associated with the active metabolism and physiology of the tissues. Notably, the limbus O2 uptake fluctuated in the day, increasing significantly in the evening, whereas the O2 uptake at the other ocular surface regions remained unchanged.In this study, we used an optical fiber sensor to examine the O2 uptake is a well-established indicator of metabolic activity, we can confidently assert that the limbus and conjunctiva had higher metabolic rates than the more central regions of the cornea. This was likely due to the limbal epithelial stem cells (LSCs) that are believed to reside at the intersection of the limbus and conjunctiva and the transiently amplifying cells that are used to replenish the epithelial cells [2 uptake, as was observed in the present study. These observations will serve as a useful baseline and framework for comparative studies with, for instance, contact lens use, ocular disease, and diabetes.Since Oal cells . Since tal cells . This tu2 influx at 5 pm. The reason for this uptick is not yet clear, but it may point to an upregulation in LSC activity in the evening. It has been seen that the murine epithelial thickness increases significantly in the evening as they become more active, peaking around 10 pm [2 uptake in mice encompassing nighttime and early morning measurements and/or utilizing a diurnal model closer to ourselves, such as a non-human primate model.Temporally, we found that in the mouse model, there was a substantial shift only in the limbus region. We observed a significant increase in the Ond 10 pm . It is pnd 10 pm . Howevernd 10 pm . We may 2 uptake with high spatial and temporal resolutions at the ocular surface. Early researchers studied O2 reduction using platinum and gold electrodes, but these were not reliable sensors due to artifacts [2 consumption due to its smaller sensor [2 that is not consumed during measurement. Combined with its self-referencing capability, O2 fluxes can be reliably and accurately measured at the ocular surface non-invasively and in vivo [2 consumption is important. This non-invasiveness and high sensitivity make the SMOT system well suited for experimental and clinical applications and therefore may well be a good standard for O2 flux measurements.This study also confirmed the micro-optrode-based SMOT system as a reliable and easily applicable technique for measuring Ortifacts . Later, r sensor ,22,23. T in vivo , thus of


Paracentrotus lividus eggs were incubated in acidic seawater, which removes the egg jelly, i.e., experimental conditions that should prevent the occurrence of the AR, and inseminated in the same medium. At variance with the prevailing view, our results have shown that these dejellied P. lividus eggs can still interact with sperm in acidic seawater, albeit with altered fertilization responses. In particular, the eggs deprived of the vitelline layer reacted with multiple sperm but with altered Ca2+ signals. The results have provided experimental evidence that the plasma membrane, and not the vitelline layer, is where the specific recognition between gametes occurs. The vitelline layer works in unfertilized eggs to prevent polyspermy.In sea urchins, the sequence of the cellular and molecular events characterizing the fertilization process has been intensively studied. We have learned that to activate the egg, the fertilizing sperm must undergo morphological modifications  upon reaching the outer gelatinous layer enveloping the egg (egg jelly), which triggers the polymerization of F-actin on the sperm head to form the acrosomal process. The AR exposes bindin, an adhesive sperm protein essential for the species-specific interaction with the cognate receptor on the egg vitelline layer. To investigate the specific roles of the egg jelly and vitelline layer at fertilization of sea urchin eggs,  Applying the formula: Finst = [Ft − F(t − 1)]/F(t − 1), the instantaneous increment of the Ca2+ level was analyzed to locate the specific area of momentary Ca2+ increase. The values of the Ca2+ signals were obtained from three independent experiments (N), and the number of eggs (n) being analyzed for each condition is specified in the Results section. To visualize the F-actin in living eggs, 10 µM (pipette concentration in methanol) of AlexaFluor 568-phalloidin (Molecular Probes) or 10 mg/mL of bacterially expressed LifeAct-GFP fusion protein were microinjected into the unfertilized eggs in two independent experiments (N). The eggs microinjected with the fluorescent actin probes were observed with a Leica TCS SP8X confocal laser scanning microscope equipped with a white light laser and hybrid detectors using the Lightning deconvolution mode . The number of eggs examined for each condition (n) is specified in the Results section.Intact sea urchin eggs were microinjected using an air pressure transjector  as previously described . To moniP. lividus eggs were counted after their visualization in the cytoplasm of fertilized eggs 5 min after insemination using a cooled CCD (Charge-Coupled Device) camera  mounted on a Zeiss Axiovert 200 microscope with a Plan-Neofluar 40X/0.75 objective and with a UV laser. The number of fertilized eggs examined (n) for each condition in two independent experiments (N) is shown in the Results section.The diluted sperm were stained with 5 µM of Hoechst-33342  for 30 s before insemination. The labeled decondensed sperm nuclei incorporated into the p < 0.05 was considered statistically significant. For the ANOVA results showing p < 0.05, the statistical significance of the difference between the two groups was assessed by Tukey’s post hoc tests. The two groups of data showing significant differences from each other were marked with symbols indicating the p values in the figure legends.The numerical MetaMorph data were compiled and analyzed with Excel (Microsoft Office 2010) and reported as mean ± standard deviation (SD) in all cases in this manuscript. A one-way ANOVA was performed through Prism 5 (GraphPad Software), and P. lividus eggs before and after the removal of the jelly and the vitelline layer. Note that the transparent egg jelly which normally surrounds the eggs was not preserved during the fixation procedure for the SEM observations. In the unfertilized control eggs (P. lividus eggs with 10 mM of DTT in NSW (pH 7.57) induced changes in the microvillar morphology and an alteration of the VL structure without removing it [P. lividus eggs were incubated in NSW containing 10 mM of DTT (pH 9) for 20 min. The SEM micrograph in The scanning and transmission electron (SEM and TEM) micrographs of rol eggs A-C, the rol eggs B, as shorol eggs D, which rol eggs E. The VLrol eggs F. The shoving it . To compP. lividus eggs fertilized in NSW (control). Five minutes after insemination, the TEM image in P. lividus eggs incubated for 5 min in acidic seawater (pH 5.5) and fertilized in the same medium is shown in P. lividus egg fertilized in NSW after 20 min of treatment with DTT in NSW at pH 9 to remove the VL and the jelly coat to which the latter is attached. The low magnification in The SEM and TEM micrographs in 2+ signals in these experimental conditions  into the eggs deprived of egg investments. nditions . Note thnditions B, it didnditions F.By contrast, the denuded eggs, after being treated with DTT for 20 min at pH 9, washed several times in NSW, and then inseminated in NSW at pH 8.1, were penetrated by multiple sperm , C possibl2+ response in sea urchin eggs is strongly linked with the microvilli morphology and structural organization of the egg cortex. In normal conditions, upon insemination, the eggs experience the first Ca2+ signal, the so-called cortical flash (CF), which takes place simultaneously at the periphery of the egg cortex as a result of the activation of the L-type Ca2+ channels promoting a Ca2+ influx. The CF is followed by a Ca2+ wave occurring during the separation of the VL from the egg surface due to the CG’s exocytosis [2+ signals at fertilization in NSW induced by two sperm in an egg deprived of the VL (and egg jelly), which in this case were elicited on the opposite sides of the egg. The graphs of 2+ increased in the control eggs fertilized in NSW at pH 8.1 (green color), eggs fertilized while incubated in acidic seawater (blue color), and eggs inseminated in NSW at pH 8.1 following the treatment with DTT for 20 min at pH 9 (red color). Both the graphs and histograms show striking differences in the pattern of the sperm-induced Ca2+ signals in the eggs inseminated in acidic seawater as judged by the remarkably delayed time required by the sperm until they initiated the CF (arrows in the graphs) as a sign of its fusion with the egg plasma membrane , which was nearly 10 times longer than that in the control eggs fertilized in NSW at pH 8.1 . When fertilized in acidic seawater, a complete inhibition of the Ca2+ response was observed in 6 out of 28 eggs; thus, in general, the sperm had difficulties initiating Ca2+ responses in the eggs incubated in acidic seawater. Even when the CF was generated, its amplitude was significantly lower  than that of the control eggs   than that of the control .Previous studies from our laboratory have amply shown that the sperm-induced Caocytosis . The rel < 0.01) C. Simila2+ responses. The graphs in 2+ increases in the stripped eggs were significantly smaller than in the control (green color). In contrast, no significant difference was detected in the time lag between the insemination and the initiation of the CF. Nevertheless, 6 out of 25 denuded eggs did not experience any CF upon insemination, possibly due to the compromised functionality of the Ca2+ influx channels as a result of the morphological changes of the microvilli where the channels reside [2+ responses in the egg, the heights of both the CF and Ca2+ wave were highly reduced in comparison with the control eggs .When the denuded eggs, i.e., deprived of both the VL and jelly as a result of the DTT treatment at pH 9, were inseminated after being washed several times in NSW at pH 8.1, they all responded to the sperm with altered Cas reside . When murol eggs C. It is P. lividus eggs were inseminated in NSW with A. lixula sperm whose DNA was stained with Hoechst 33342 to eventually visualize them inside the eggs. The capability of the heterologous sperm of A. lixula to transduce its Ca2+ signals was also tested. The fluorescent and transmitted light images of P. lividus eggs were cross-inseminated with labeled A. lixula sperm, the sperm were seen to be embedded in the jelly layer surrounding the eggs and around the egg surfaces. The graph of 2+ signals that were induced following the insemination of whole P. lividus eggs with A. lixula sperm. An analysis of the Ca2+ changes in the egg as a result of the fusion between the plasma membranes of the two gametes showed that out of 17 eggs, only three responded to the sperm with a CF of a smaller amplitude as compared to that elicited by homologous sperm  and smaller CF elicited in 18 out of 26 denuded eggs upon the addition of A. lixula sperm .At variance with the intact eggs, when denuded membrane B. A compP. lividus eggs by several agents compromised the translocation of actin fibers following the fertilization of P. lividus eggs [P. lividus eggs [P. lividus eggs in normal and acidic seawater experimental conditions. Five minutes after the sperm addition, a dense actin layer was visualized in the cortical region. A spike formation in the perivitelline space (PS) accompanying the elevation of the FE was also visible 20 min after insemination (n = 13) in the overlay image. Even if it was still possible to visualize a layer of actin in the eggs incubated and fertilized in acidic seawater, an evident inhibition of the polymerization of actin was shown in the confocal image captured 5 min after insemination. Actin polymerization, albeit much thinner than in the control eggs, was manifested 20 min after the insemination of eggs in acidic seawater, showing an impaired elevation in the FE (n = 6). Another sign of the alteration of the egg surface by the acidic treatment was the presence of sperm attached to the “sticky” egg surface 20 min after insemination, usually not seen in the control eggs at fertilization  has been well characterized in vitro and in vivo [2+ increases in the egg cortex when the head of the fertilizing sperm was outside the egg jelly [The initial act in the chain of reactions during the fertilization process is the interaction between the fertilizing sperm and the egg surface. This ultimately leads to the union of the chromosomes carried by the sperm and the egg pronucleus. Information on the molecular mechanism regulating the sperm–egg interaction has been mainly based on research on marine animal models, such as sea urchins and starfish, that reproduce by external fertilization. To ensure monospermic fertilization, sea urchin eggs seem to operate both a fast electrical block to polyspermy, which starts as soon as the sperm fuses with the plasma membrane  and a memediated , as initmediated ,40. In aseawater ,41,42,43seawater . In this in vivo ,45,46. T in vivo , and by gg jelly ,49,50. Agg jelly ,52. Furtgg jelly . In this2+ signal following sperm–egg membranes’ fusion in control and dejellied eggs suggested that neither the egg jelly nor the VL is the site at which P. lividus sperm undergo the AR. Indeed, the results of this contribution have shown that the sperm are still able to fertilize the eggs incubated in acidic seawater, which should prevent the polymerization of the actin of the acrosomal process, known to be induced by a pH increase. A considerable time delay between the addition of the sperm and the onset of the first Ca2+ is attributable to microvillar morphology changes and shrinkage of the egg, causing an alteration of the sperm-induced Ca2+ signals (2+ response at fertilization of this sea urchin species [P. lividus eggs in seawater titrated to pH 9 by NH4OH [2+ response upon insemination (data not shown).A critical comparative analysis of data from the literature on the adaptive variety of the molecular mechanisms regulating the sperm–egg interaction in the course of evolution led to the suggestion that, as with mammalian and ascidians’ eggs, sea urchin eggs also have the VL and not the egg jelly as the site at which the fertilizing sperm undergoes the AR ,55,56. T species ,31,32,33by NH4OH . These f2+ signals . The morphological modifications of the egg surface promote the fusion of multiple sperm, generating impaired Ca species B. Thus, P. lividus sperm to the egg plasma membrane occurs at a preferential site uncovered by the vitelline layer. An explanation that sounds plausible for the recent finding in starfish is that the extension of the long acrosomal filament is formed to span the thickness of the egg jelly to reach and fuse with the egg plasma membrane through pre-existing openings on the VL [P. lividus is achieved when the structural integrity of the VL of the unfertilized eggs is altered [Our observations may suggest that, at fertilization, the binding and fusion of the n the VL ,49,50. Tn the VL . It foll altered ,34 or coDespite the significant structural diversity of eggs and sperm of the animal species and the different physiological behaviors, the fundamental event of the fertilization process, which depends on species-specific recognition, is the fusion of the sperm with the egg plasma membrane to transduce the fertilization signals. At the fertilization of sea urchin eggs, the species-specific molecular events regulating the gamete interactions have highlighted the crucial role of the sperm receptors on the vitelline layer for binding the acrosome-reacted sperm exposing the adhesive protein, bindin, for fusion with the egg plasma membrane. At variance with the prevailing view in the literature, the results of this contribution have shown that restricted regions of the egg plasma membrane are differentiated to fuse with the plasma membrane of acrosome-unreacted homologous, and not heterologous, sperm. Thus, the integrity of the structure of the vitelline layer covering the surface of unfertilized eggs is essential to mask the fusogenic sites residing in the plasma membrane, the exposure of which would induce polyspermic homologous fertilization. Our results on the requirement of specific receptors on the plasma membrane, and not the vitelline layer, for the specific recognition and fusion with sperm may help us to understand the role played by the vitelline layer in the eggs of different species to seek common basic molecular mechanisms regulating fertilization in other animal groups.