The objective of this study was to compare long-latency auditory evoked potentials before and after hearing aid fittings in children with sensorineural hearing loss compared with age-matched children with normal hearing.Thirty-two subjects of both genders aged 7 to 12 years participated in this study and were divided into two groups as follows: 14 children with normal hearing were assigned to the control group (mean age 9 years and 8 months), and 18 children with mild to moderate symmetrical bilateral sensorineural hearing loss were assigned to the study group (mean age 9 years and 2 months). The children underwent tympanometry, pure tone and speech audiometry and long-latency auditory evoked potential testing with speech and tone burst stimuli. The groups were assessed at three time points.The study group had a lower percentage of positive responses, lower P1-N1 and P2-N2 amplitudes (speech and tone burst), and increased latencies for the P1 and P300 components following the tone burst stimuli. They also showed improvements in long-latency auditory evoked potentials (with regard to both the amplitude and presence of responses) after hearing aid use.Alterations in the central auditory pathways can be identified using P1-N1 and P2-N2 amplitude components, and the presence of these components increases after a short period of auditory stimulation (hearing aid use). These findings emphasize the importance of using these amplitude components to monitor the neuroplasticity of the central auditory nervous system in hearing aid users. Hearing thresholds obtained using behavioral tests do not provide comprehensive data regarding the contribution of individual sound amplification devices (hearing aids [HAs]) and/or cochlear implants to the central auditory nervous system. For this reason, the use of objective tests is essential Currently, long-latency auditory evoked potentials (LLAEPs) are used to investigate impairments in the central auditory pathways of children LLAEPs are composed of the P1, N1, P2, N2 and P300 components. P1, N1 and P2 are considered exogenous components, i.e., they are influenced by the acoustic characteristics of a stimulus, while the N2 and P300 components are endogenous, i.e., they are most influenced by intrinsic events such as perception and cognition LLAEPs can be obtained using different acoustic stimuli (pure tones or speech), and responses to tonal stimuli have lower latency than those obtained from a speech stimulus Impairment of the LLAEP components P1, N1, P2, N2) in children with mild to moderate SNHL has been reported in a previous study, which showed that this population has a deficit in central auditory processing , N2 in cAccording to the literature, the presence of the P1, N1, P2 and N2 components using speech and tone burst stimuli in children with SNHL is positively correlated with the duration of HA use, and the latency of the N1 and P2 components is positively correlated with both the duration of HA use and the child’s age The P300 component is present among people with hearing loss as long as the individual can perceive and distinguish acoustic stimuli Individuals (children and adults) with severe or profound congenital hearing loss showed 58.6% presence of P300. Individuals with profound hearing loss showed lower amplitudes than those with severe hearing loss, but there were no differences in latency Given the importance of proper central auditory nervous system functioning in childhood development, the aim of this study was to provide scientific evidence for the development and plasticity of the central auditory nervous system in response to HA use using LLAEPs. The study will evaluate the effectiveness and benefits of sound amplification (stimulation) in hearing-impaired children. This study compares LLAEPs in children with SNHL before and after HA fittings compared with those in age-matched children with normal hearing.This was a longitudinal prospective clinical study approved by the Ethics Committee for Research Project Analysis under Protocol No 266512/2013. The participants’ legal guardians read and signed the Terms of Free and Informed Consent (TFIC) form, and the children signed the Terms of Assent form.A total of 32 subjects of both genders aged 7 to 12 years participated in this study. The participants were divided into two groups as follows: 14 comprised the control group , and 18 comprised the study group .The CG comprised children who had normal otoscopy findings, auditory thresholds up to 15 dB HL (frequencies of 250 to 8000 Hz), a type A tympanometric curve The SG comprised children who had normal otoscopy findings, mild to moderate bilateral symmetrical and flat SNHL ). All children with hearing loss were referred to the Auditory Health Clinic of the Clinics Hospital by the Central System for Regularization of Health Services Offering  of the State Health Department of São Paulo. Due to the above referral system, at the time of the audiological and electrophysiological hearing assessments, none of the children had undergone any type of rehabilitation for their hearing loss.Hearing loss in the SG was diagnosed at the Hearing Clinic of the Clinical Hospital of the School Medicine of the University of São Paulo - Division of Clinical Otorhinolaryngology , suspected prematurity in 5.55% 5, suspecAfter the children were diagnosed with hearing loss, they were referred for electrophysiological evaluation of hearing. At a later date, they returned to the outpatient clinic for selection and fitting of HAs.In all cases, the HA was a bilateral mini retroauricular-type device with digital technology and non-linear signal processing. All children with mild or moderate hearing loss underwent a HA fitting. The children used their HAs daily, and the daily use was monitored through the processing algorithm that is built into the HA’s memory (mean time of 8.5 hours of daily use).The children underwent tonal and vocal audiometry using a Grason Stadler GSI 61 audiometer and a TDH 50 supra-aural earphone, acoustic emittance measurements using a Madsen Model Zodiac 901 emittance meter, and LLAEP examination using a two-channel device  calibrated at listening level (dBHL). The acoustic stimulation used to acquire the LLAEPs was presented by a sound field system with speakers positioned at a 90-degree angle and 45 cm from the ear to be tested.Both groups were assessed at three different time points: baseline (M0), 3 months after the initial assessment (M3), and 9 months after the initial assessment (M9). The SG children did not use HAs at M0.The LLAEPs were obtained by the same evaluator at the three time points. The components were analyzed by the evaluator and two other professionals with experience in hearing electrophysiology using consensus scoring.To capture the P1, N1, P2, N2 and P300 components of the LLAEPs, the child was placed in a reclining chair in an acoustically treated room with appropriate electrical access. The skin was cleaned with abrasive paste, and the electrodes were attached to the skin with electrolytic paste and adhesive tape (Micropore) at the active vertex (Cz) and ground (Fpz) based on the International Electrode System standard IES 10-20 LLAEPs were evoked by tone burst stimuli using 1000 Hz as the frequent stimulus and 2000 Hz as the rare stimulus. The stimuli were delivered for 50 ms at 75 dBnHL at a display speed of 1.1 stimuli per second using an exact Blackman envelope and an interstimulus interval (ISI) of 860 ms. We chose the frequencies between 1000 and 2000 Hz based on the literature and used the protocol proposed by Hall, 2007.For the speech stimuli, the /ba/ syllable was used as the frequent stimulus and the /da/ syllable was used as the rare stimulus. The stimuli were presented at 75 dBnHL at a presentation rate of 1.1 stimuli per second. The frequent stimulus (/ba/) had a duration of 114 ms and an ISI of 799 ms, while the rare stimulus (/da/) had a duration of 206 ms and an ISI of 690 ms. Both stimuli were synthetic. The specific characteristics of the /ba/ and /da/ speech sounds are described in A total of 300 stimuli (15% rare and 85% frequent) were used to capture the P300 component. The children were instructed to pay attention to the rare stimuli, which occurred randomly among a series of frequent stimuli, and to raise their hand whenever the rare event occurred.During data collection, responses were recorded on two charts: one chart corresponded to the frequent stimulus and identified and analyzed the P1, N1, P2 and N2 components, and another chart corresponded to the rare stimulus and identified and analyzed the P300 component. The amplitude and latency of all components and the presence and absence of responses for each ear were analyzed.Each child was instructed to look at a fixed point two meters in front of him/her. The charts were accepted for analysis when a maximum of 30 artifacts were present.In addition to the tabulation of the latency and amplitude values of the LLAEP components at each assessment point, the LLAEP components were classified as present or absent for each individual and for each studied ear.First, the percentages of present and absent LLAEP components were calculated for both groups. We compared the latency and amplitude values within each group and between groups at each time point .The Shapiro-Wilk and Levene tests were performed to verify that the groups had a normal distribution and to determine whether there was homogeneity of variances between groups, respectively.p value ≤0.05) for all analyses.Descriptive analyses were performed using means and standard deviations (± SD). When comparing three values, the three-factor repeated measures analysis of variance was used. When comparing two values, the two-factor analysis or the Bonferroni test was used. The significance level was set at 5%  in the P1-N1 amplitude value in the right ear (RE), and the SG presented a lower mean amplitude at all assessment points , there was a statistically significant difference (t points .p=0.003), P2-N2 in the LE at M3 (p=0.032) and P300 at M0 (p=0.010)  .p=0.027; M0xM9 p=0.060) and in the P300 amplitude in the LE   .The comparison of the CG and SG at the three time points and the two-factor comparison revealed no statistically significant difference in the latency of the LLAEP components .p=0.007; M3XM9 p=0.010) and N2  were found for the LE in the SG. In the CG, a statistically significant increase in the P2 latency  in the LE was observed , statistically significant differences in the P1-N1 amplitude were found in both ears, and the SG had lower amplitudes at all time points .p=0.008) and in the LE at M1 (p=0.036). We also observed significant differences in P2-N2 in the RE at M3 (p=0.039) and M9 (p=0.019)  . For botComparisons between the time points revealed no statistically significant differences between the P1-N1, P2, N2 and P300 amplitudes in either the CG or SG .p=0.013) in the LE, with the SG showing a greater latency value.The comparison of the control and study groups at the three time points revealed no significant difference in the latency of the LLAEP components. In the two-factor comparison, we observed a significant difference only for P300 at M0  and P1 in the LE  and P300 in the RE  in the SG  components are influenced by the physical characteristics of the stimulus The comparison of the P300 amplitudes in both the CG and SG  and 5 reThese findings are similar to those reported in a study showing that P300 was present in 100% of subjects with normal hearing or mild to moderate SNHL at an intensity of 80 dB SPL Furthermore, although the difference was not statistically significant, the SG showed an improvement in the P300 amplitude between M0 and M3 for both stimulus types. These results suggest possible neuroplasticity due to HA use. According to the literature, neuroplasticity occurs as a result of the nervous system’s ability to reorganize its structures, functions and connections in response to intrinsic and extrinsic stimuli According to the literature In this study, the comparison of responses to the speech stimulus between the CG and SG  and 3 shThe results of this study show that, for most LLAEP components, the SG and CG had equivalent acoustic stimulus transmission/processing speeds. These findings may be related to the SG’s degree of hearing loss. It is noteworthy that in mild/moderate recruitment hearing loss, the impairment may reside in the outer hair cells (OHCs) Another possible cause of the similar latency found in both groups may be related to plasticity resulting from auditory deprivation, i.e., the central nervous system reorganizes to compensate for a peripheral receptor disorder Although there were no differences in latency between the two groups, the intra-group comparisons at the different time points revealed some differences  and 4. TThe results show that after three months of HA use, there were improvements in the response generation times of the P2 and N2 (speech stimulus) and P1 and P300 (tone burst stimulus) components, likely due to neuroplasticity in response to auditory stimulation (HA use).These findings are in accordance with a previous study There are some limitations to the current study, such as the number of individuals in the sample. The number of participants in the SG was variable at different time points due to the longitudinal design, which depended on the compliance of patients for audiological follow-up. For this reason, it was not possible to achieve an equal number of individuals at the three time points for audiological follow-up. In addition, it was not possible to obtain a homogeneous sample with consistent etiology and hearing loss onset. Regarding the control group, we believe that the low number of individuals may be related to the fact that they were typical developed children, with no hearing complaints.In conclusion, the results of this study demonstrated reductions in amplitudes of the P1-N1 and P2-N2 components of the LLAEP and a greater percentage of absent responses in children with mild to moderate SNHL, suggesting a deficit in the activation of the cortical structures responsible for auditory processing of the exogenous characteristics of these stimuli.P300 showed similar responses between the groups, suggesting that the endogenous potential was less affected than the exogenous potential by the sensory input deficiency caused by the hearing loss in this population.The latency of the LLAEP components showed little variability between the groups, indicating that sensory hearing loss did not interfere with the stimulus processing speed. The responses were steady across the three time points, demonstrating that this measurement is minimally affected over a short time interval (9 months).The results of the present study show that it is possible to identify changes in the central auditory pathway using P1-N1 and P2-N2 component amplitudes and that there is an increased response of these components to a short period of auditory stimulation . Therefore, the importance of using these components to monitor the neuroplasticity of the central auditory nervous system in HA users should be emphasized.Leite RA participated in the conception and design of the study, acquisition, analysis and interpretation of the data, drafting and revision of the manuscript for important intellectual content, and approval of the final version of the manuscript to be published. Magliaro FC participated in the conception and design of the study, acquisition, analysis and interpretation of the data, drafting and revision of the manuscript for important intellectual content, and approval of the final version of the manuscript to be published. Matas CG participated in the conception and design of the study, the analysis and interpretation of the data, drafting and revision of the manuscript for important intellectual content, and approval of the final version of the manuscript to be published. Raimundo JC participated in the conception and design of the study, acquisition of data, and critical revision of the manuscript for important intellectual content. Bento RF participated in critically revising the manuscript for important intellectual content.


Increasing amounts of evidence support the role of inflammation in epilepsy. This study was done to evaluate serum follow-up of IL-1β and IL-6 levels, as well as their concentration in the neocortex, and the relationship of central inflammation with NF-κB and annexin V in drug-resistant temporal lobe epileptic (DRTLE) patients submitted to surgical treatment. Peripheral and central levels of IL-1β and IL-6were measured by ELISA in 10 DRTLE patients. The sera from patients were taken before surgery, and 12 and 24 months after surgical treatment. The neocortical expression of NF-κB was evaluated by western blotting and annexin V co-localization with synaptophysin by immunohistochemistry. The neocortical tissues from five patients who died by non-neurological causes were used as control. Decreased serum levels of IL-1 and IL-6 were observed after surgery; at this time, 70% of patients were seizure-free. No values of IL-1 and IL-6 were detected in neocortical control tissue, whereas cytokine levels were evidenced in DRTLE. Increased NF-κB neocortex expression was found and the positive annexin V neurons were more obvious in the DRTLE tissue, correlating with IL-6 levels. The follow-up study confirmed that the inflammatory alterations disappeared one year after surgery, when the majority of patients were seizure-free, and the apoptotic death process correlated with inflammation. Epilepsy is a chronic brain disease that affects around 50 million people worldwide. In a high number of cases it is characterized by the presence of recurrent seizures, which are the result of an excessive electrical discharge of a neuronal group in a certain part of the brain . The batSeveral authors suggest the contribution of immunological and inflammatory mechanisms in the pathogenesis of epilepsy based on the favorable effects of treatment with intravenous immunoglobulins, corticosteroids, and anti-inflammatories ,8,9,10. Inflammation is considered an important factor in the pathophysiology of seizures. All the risk factors for epilepsy such as traumas, tumors, and infections are accompanied by different degrees of inflammation in the central nervous system, which is associated with the occurrence of seizures ,18,19. HProinflammatory cytokines are highly studied markers in both patients and experimental models of drug-resistant epilepsy. IL-1 and IL-6 are two of the most commonly approached proinflammatory cytokines in studies conducted to evaluate inflammation in drug-resistant epilepsy ,24,25,26The main objective of this work is to evaluate the proinflammatory proteins after the elimination of the epileptogenic focus and its relation with the neuronal loss described in patients with DRTLE.Ten patients from the International Center for Neurological Restoration (CIREN) with DRTLE  who underwent epilepsy surgery were included for the study of serum concentration of inflammatory cytokines (IL-1β and IL-6). The criteria to be considered drug-resistant were the following: present seizures for more than two years, two complex partial monthly seizures, use of two first-line anti-epileptic drugs, two cycles of monotherapy and at least one of polytherapy.All patients were evaluated in the Video-EEG Telemetry Unit from CIREN, and they underwent a complete general and neurological physical examination and anatomical evaluation by Magnetic Resonance Imaging (1.5 T MAGNETOM SINPHONY) and SPECT.Temporal localization presents unilateral rhythmic unloading with maximum amplitude in the zygomatic electrodes and in the anterior or middle temporal ones as the first electrographic change.Antiepileptic treatment: The most commonly used drug was Carbamazepine (3–6 tablets daily) followed by Clobazan (3–4 tablets daily), Magnesium Valproate (1–6 tablets daily), and Lamotrigine (2–3 tablets daily). Up to two years after the surgery, all patients received the same drug treatment.The neocortex adjacent to the hippocampus of the patients was resected at the time of surgery and the resection of the epileptic zone was performed by means of standard temporal lobectomies adjusted by electrocorticography.The neocortical tissues from patients in this study were evaluated to determine the concentrations of IL-1β and IL-6, co-localization of annexinV and synaptophysine, and the expression of NF-κB. The neocortex control tissues were obtained from subjects who died due to non-neurological causes . The control tissues were matched in age and gender with the group of DRTLE patients . They did not show macro- or microscopic pathological abnormalities, and the mean time of obtaining tissue was 3.2 h after the subject died.The collected clinical parameters included demographic characteristics and clinical state showed in Blood: Venous Blood samples (5 mL) of the patients were taken by antecubital puncture, after asepsis of the region. The blood was centrifuged and the serum was kept at −20 °C until use. The samples of serum were obtained before and after surgical treatment .3 was cut, placed in liquid nitrogen, and later transferred to a −80 °C freezer where it was preserved until use.Neocortical tissue: The neocortical tissue from patients with DRTLE was obtained during surgery. The tissue was washed with 0.9% saline solution at 4 °C, a fragment of approximately 0.5 cm3 was taken from the neocortex; this fragment was processed in a similar way to patient’s tissues.The neocortical control tissue was obtained from necropsy and was subsequently fractionated by areas. A fragment of approximately 0.5 cmn = 4) and pulmonary thromboembolism (n = 1), and without history of neurological disease (Control samples were obtained from five subjects who died by vehicle accident ( disease . The neoHomogenization of brain tissue: Tissue samples were homogenized in a glass–Teflon (Potter-Elvehjem), containing lysis buffer as described by the manufacturer for the cytokine ELISA and 1mL of Trizol reagent  for the western blot. Protein was isolated following the manufacturer’s instructions.2SO4 and optical densities were measured at 450 nm using a microplate reader .The concentrations of IL-1β and IL-6  were measured by ELISA according to manufacturer’s instructions. The concentrations of IL-1β and IL-6 from the homogenate of the neocortical tissue of patients with DRTLE and control subjects was measured by the same methods. The lower limit of detection was 6pg/mL for IL-6 and 1 pg/mL for IL-β. Briefly, serum or homogenate from neocortical tissue was incubated in coated 96-well plates at room temperature for 2 h. The serum and tissue samples were applied in duplicate as well as the dilutions from the standard curves (IL-1β and IL-6). Plates were washed and then incubated with the detection antibody. After rewashing the plates, the conjugate was added for 30 min, followed by the substrate solution. The reaction was stopped with 1N HThe expression levels of NF-κB in the homogenates were analyzed by gel electrophoresis and western blot. The concentration of total proteins was determined by Lowry's method . The absTo determine the integrity of the extracted proteins, 1D SDS-PAGE electrophoresis (12%) was used, followed by staining with Coomassie blue and western blot to identify the constitutive protein, indicating that there is no protein degradation.The fragment of neocortical tissue obtained during surgery was washed with 0.9% saline at 4 °C and fixed in phosphate solution containing 4% paraformaldehyde and 0.2% glutaraldehyde. Subsequently, they were submerged in increasing solutions of sucrose  and frozen at −70 °C. The neocortex tissue was cut into sections of 10 μm . The pieces of tissue were blocked and incubated with the apoptotic marker annexin V and neuronal marker synaptophysin  according to the instructions of the manufacturer. An antibody conjugated with FITC  was used for the immunodetection of antibodies against annexin V, while the immunodetection of synaptophysin was carried out by means of an antibody conjugated with Alexa Fluor 647 . The co-localization of the apoptotic and neuronal markers was evaluated in each patient’s tissue. The nuclei were visualized by counterstaining with propidium iodide. All sections were examined in a confocal microscope  and each plate was examined by two specialists (SO and LL).3) was calculated in relation to the number of cells stained with propidium iodide per mm3, and the result was expressed as the percentage of cells positive for each marker. All sheets were examined by two specialists (SO and LL).Quantitative Analysis: Only the sections that had consistent immunostaining were counted and 5 to 10 sections of each patient or control were quantified. The percentage of immunopositive cells to each marker . The values are expressed as mean ± SEM. Normal distribution and homogeneity of variance of the data were tested by the Kolmogorov–Smirnov and Levene tests, respectively. The comparisons between two groups were made by means of the In order to evaluate the effect on serum concentrations of IL-1β and IL-6 in DRTLE patients who received surgical treatment, we measured the concentrations of these cytokines before, and one and two years after the resection of the epileptogenic zone. In this study, there are no gender differences inboth levels of interleukins (IL-1 and IL-6), so the concentrations in male and female sexes were unified in one group. The results showed a statistically significant decrease of both (IL-1β and IL-6) cytokines A,Bone anNeocortical tissue from DRTLE patients showed values of IL-1β between 9.69 and 78.32 pg/mL with a mean value of 21.58 pg/mL A, and ILThe presence of the molecule involved in the inflammatory cascade and linked to death processes in neocortical tissue, NF-κB, was evaluated, and the results are shown in There is a positive correlation between the percentage of immunopositive cells to annexin V and the concentration of IL-6 in neocortical tissue .The concept of “immunological privilege” of the CNS has been eliminated because of, among other reasons, the accumulation of evidence that immune responses and inflammatory reactions are involved in the pathogenesis of several brain diseases like epilepsy . There iThe study of different models of epilepsy developed to evaluate the inflammatory response has shown that, in rodents, with convulsive activity occurs the following molecular cascade: a rapid increase in the proinflammatory cytokines , the activation of the signaling pathway of Toll-like receptors (TLRs), the production of chemokines, activation of the complement system, and increased expression of adhesion molecules ,37,38,39On the other hand, the literature supports that the immunological and inflammatory alterations described in epilepsy may be associated with anti-epileptic drugs. Changes in inflammatory markers related to carbamazepine, valproate, and other drugs are described ,41.Serum levels of IL-1β and IL-6 before and after surgery were evaluated in order to detect if there were changes in the peripheral proinflammatory cytokines from DRTLE patients and if these changes were modified once the epileptogenic zone was resected. This follow-up study was related to the clinical evolution of these patients in terms of the presence or absence of seizures one and two years after surgery. The decrease in the concentration of IL-1β and IL-6 one year after surgery and the fact that in this period most of the patients are free of seizures supports the idea that seizures are the cause of the inflammatory disorders observed in patients with DRTLE. Interestingly, 70% of patients remained seizure-free one year after surgery and the remainder (25%) showed a substantial reduction in seizure frequency.According to our results, the low levels of IL-6 in patients who were free of seizures one year after surgery in comparison with those in the patients who still experienced them allow us to confirm that this inflammatory marker is closely linked with the occurrence of seizures. Our results indicate that once the epileptogenic zone is resected and seizure activity is reduced, there is a decrease in proinflammatory cytokines. These findings may suggest that seizures are the cause of inflammatory disorders observed in patients with DRTLE.Other authors report an increase of IL-6 concentrations in patients with epilepsy compared with those in control subjects. They describe this increase associated with the temporal location of the epileptogenic zone . CoincidAn important aspect to assess is the integration of inflammatory events that occur both peripherally and centrally. The existence of several mechanisms by which peripheral inflammation interacts with the brain and induces inflammation at the central level has been discussed. The immediate recognition of inflammatory mediators such as cytokines can occur in circumventricular organs due to the expression of TLRs and IL-R in these structures . The cytThe evaluation of IL-1β and IL-6 levels in brain tissue (neocortex) of patients with DRTLE showed values that indicate the presence of an inflammatory process at the central level reinforced by the absence of these markers in the control tissue. Tissue obtained at autopsy from subjects with no evidence of neurological disease was used as controls since previous reports indicate that proteins from brain tissue are preserved for several hours after death ,29,30,49In particular, the analysis of the mRNA of IL-1β and IL-1Ra after the systemic injection of kainic acid in rats shows a significant induction in the microglial cells of the hippocampus, as well as in other areas of the limbic system . VezzaniIndistinctly in blood or cerebrospinal fluid, an increase in IL-1β and its receptor in astrocytes, microglia, and neurons was documented in a series of patients surgically treated for TLE with hippocampal sclerosis. IL-1β and its receptor are highly expressed in the neurons and glia of patients with TLE caused by focal cortical dysplasia and neuroglial tumors, where expression in brain tissue without these alterations is negligible .In summary, we can affirm that there are high IL-1βand IL-6 concentrations at both the systemic and the central levels that sustain the presence of an inflammatory response in DRTLE. The decrease of both cytokines after surgery in patients with a drastic reduction in seizures supports the idea that these events are an important cause of inflammation.Previous studies report neuronal death in the neocortical tissue of patients with DRTLE ,57, makiIncreased expression of NF-κB in the neocortical tissue of patients with DRTLE was observed. This fact confirms the activation of the inflammatory response at the central level and helps to explain the presence of apoptotic neuronal death observed in this study.NF-κB is a key mediator of the innate and inflammatory immune response; it is activated by inflammatory cytokines through the TNF receptor, IL-1 receptor, and the TLRs family, and, in turn, their activation gives rise to the expression of other cytokines, proteases, and metabolic enzymes .NF-κB has been shown to be involved in the neuropathological processes associated with seizures in epilepsy. Epileptic seizures have been shown to trigger a rapid increase in the amount of NF-κB activated in the hippocampus  and its There are contradictory criteria related to the existence or otherwise of other affected areas different to the mesial regions in DRTLE . PreviouAnnexin V is described as an early marker of apoptotic processes. Once the process of apoptosis in a cell is activated, the externalization of phosphatidyl serine that is distributed asymmetrically in the cell membrane is produced and is able to bind annexin V. In our study, increased immunoreaction to coincidental annexin V was detected with immunodetection to synaptophysin in the neocortex of patients with DRTLE, which confirms the presence of neurons that initiate the apoptotic process in this tissue.The neuronal death observed in our results is probably due to the inflammatory process and to the involvement or mitochondrial dysfunction caused by the depolarization of the membrane and the loss of permeability of the mitochondrial membrane, a fact that usually implies the death of the cell . ProlongThe relevant role of apoptotic death in epilepsy has been affirmed by other groups ,66,67. SIn summary, our results show the increase in immunodetection with annexin V and the existence of a correlation between the concentration of IL-6 and the percentage of annexin V positive neurons in patients with DRTLE, which speaks in favor of the occurrence of an apoptotic neuronal death process. Similarly, we have shown an increase in the expression of NF-kBas well as the cytokines IL-1β and IL-6, and immunodetection of annexin V co-expressed with positive synaptophysin cells. All these findings support the occurrence of inflammatory and apoptotic processes in DRTLE patients.The evaluation of the participation of inflammatory processes in DRTLE is aimed at answering one basic question: Is inflammation part of the etiopathogenic processes in DRTLE or are the seizures the result of inflammatory disorders? In this sense, our results describe the clinical evidence that once the epileptogenic tissue has been resected with consequent elimination of seizures (or a significant reduction of them), the observed inflammation disappears. This finding supports the hypothesis that the inflammation could be mostly the consequence of epileptogenic processes. Similarly, this study supports the criterion of the involvement of inflammation and the activation of pathways such as NF-κB, and the final occurrence of death processes of the neuronal population is represented.All these results highlight the necessity for new research about the role of inflammation and the immune response in the CNS, particularly in DRTLE, in order to achieve an understanding of the epileptogenic mechanism in this clinical entity and open up new possible immunomodulator treatments, in particular for those cases in which surgery is not a therapeutic alternative.


In this work, we review the state of the art in G6PD deficiency, describing 217 mutations in the g6pd gene; we also compile information about 31 new mutations, 16 that were not recognized and 15 more that have recently been reported. In order to get a better picture of the effects of new described mutations in g6pd gene, we locate the point mutations in the solved three-dimensional structure of the human G6PD protein. We found that class I mutations have the most deleterious effects on the structure and stability of the protein.Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme in the pentose phosphate pathway which produces nicotinamide adenine dinucleotide phosphate (NADPH) to maintain an adequate reducing environment in the cells and is especially important in red blood cells (RBC). Given its central role in the regulation of redox state, it is understandable that mutations in the gene encoding G6PD can cause deficiency of the protein activity leading to clinical manifestations such as neonatal jaundice and acute hemolytic anemia. Recently, an extensive review has been published about variants in the  Class I mutations more frequently affect the exons 6, 8, 10, and 13 encoding the regions that bind the enzyme substrate, dimer interface, and NADP+ structural site, respectively [The analysis of Class I G6PD mutants using the X-ray structure  showed tectively ,63,64,65The clinical manifestation of G6PD deficiency in humans has a broad clinical spectrum ranging from almost asymptomatic individuals to those with severe neonatal jaundice, acute hemolytic episodes, and chronic non-spherocytic hemolytic anemia suggesting that gene-environment interactions may influence the clinical outcome of G6PD deficiency ,67,68. TThe most severe clinical symptom of G6PD deficiency is neonatal jaundice (NNJ), which peaks two to three days after birth . In newbThe acute hemolytic anemia (AHA) is the most common manifestation of the deficiency, which is originated when the RBCs are under oxidative stress and may be triggered by a range of exogenous agents as fava beans, drugs, or infections, causing intravascular hemolysis and jaundice . The mosVicia faba) and its pathological effects (Favism) had been observed for centuries in the Mediterranean countries [The relationship between the ingestion of dry grains or frozen fava bean  as G6PD Zacatecas , HamburgD enzyme ,63,64,65We are updating 16 new mutations that have not been considered in the most recent review and 15 mutations that have been recently described. Most of these variants (11) are considered as Class I, which is the most severe symptomatic group. We localize several of these mutations using bioinformatics tools in the solved three-dimensional structure of the human G6PD protein. The mutations have been found mainly in the coding regions and are buried in the enzyme; some of these are close to active- and NADPH-binding sites affecting the structure and function of this essential enzyme and leading to a diminution in the efficiency of this essential metabolic enzyme, equivalent to the changes to its own structure and function. The G6PD deficiency produces a wide variety of clinical manifestations, with severe hemolytic anemia being one of the most dangerous. This deficiency is caused by a great variety of mutations, with single nucleotide substitutions being the most frequently described followed by multiple mutations, deletions, and intron’s mutations. The characterization of the mutations responsible for G6PD deficiency, give us an integral vision of the disease and open new possibilities for an opportune detection and treatment of this deficiency.


The results are compared to other published results. The results of these simulations achieve minimum power consumption, less complicated setup, and high collecting efficiency. All simulation programs are built by MATLAB.The separation of blood components  is important for medical applications. Recently, standing surface acoustic wave (SSAW) microfluidic devices are used for the separation of particles. In this paper, the design analysis of SSAW microfluidics is presented. Also, the analysis of SSAW force with Rayleigh angle effect and its attenuation in liquid-loaded substrate, viscous drag force, hydrodynamic force, and diffusion force are explained and analyzed. The analyses are provided for selecting the piezoelectric material, width of the main microchannel, working area of SAW, wavelength, minimum input power required for the separation process, and widths of outlet collecting microchannels. The design analysis of SSAW microfluidics is provided for determining the minimum input power required for the separation process with appropriated the displacement contrast of the particles.The analyses are applied for simulation the separation of blood components. The piezoelectric material, width of the main microchannel, working area of SAW, wavelength, and minimum input power required for the separation process are selected as LiNbO White blood cells (WBCs), red blood cells (RBCs), and platelets are the components of blood. The separation of blood components is important for medical treatment and diagnosis. The standing surface acoustic wave (SSAW) microfluidic devices are positively significant in the separation of particles especially in biomedical applications. The separation of particles is depending on volume, density, and compressibility of the particles. The separation of particles using SSAW microfluidic devices has many advantages such as: miniaturization, high accuracy, high sensitivity, portable, low cost, low time consumption, and low samples consumption.d is the finger width of IDTs. The acoustic wave propagation velocity is determined by the acoustic mode, orientation and the type of substrate. The fundamental resonant frequency is related to the acoustic wave velocity as given as following:f0, v0, and λ are the unperturbed frequency, the unperturbed velocity and wavelength of SAW, respectively. The interaction between SAWs and the medium can be used as sensors [The surface acoustic wave (SAW) is generated by applying alternating electric (AC) signal to interdigital transducers (IDTs) based on the piezoelectricity phenomenon. IDTs are metal electrodes deposited on the piezoelectric substrate. The acoustic waves are launched and propagated away from and perpendicular to IDTs fingers. The wavelength is determined by the IDT geometry as following :λ = 4d without using sheath flow was demonstrated. The applied input power was ranged from 320 mW to 1280 mW and the flow rate ranged from 0.2 μL/min to 2 μL/min. The separation efficiencies obtained were in the range of 98.8%–89.4%for flow rates ranging from 0.2 μL/min to 2.0 μL/min at 1280 mW input power.In Reference , the sep3 and 1710 kg/m3) was demonstrated. The applied input power was ranged from 300 mW to 1200 mW and the flow rate ranged from 0.1 μL/min to 0.5 μL/min. The captured fluorescent images were used to verify the experimental results.In Reference , the sepIn Reference , input pE. coli bacteria were demonstrated. The first sample was a mixture of two types of fluorescent microspheres, 1.2 μm in diameter with green emission and 5.86 μm in diameter with red emission. The applied input powers were 85 mW, 240 mW, and 339 mW. The Flow rates of the particle flow and a single side sheath flow were 0.2 μL/min and 0.8 μL/min, respectively. The input power 85 mW was weak to produce sufficient acoustic radiation force. On the other hand, the input power 339 mW was strong to produce sufficient acoustic radiation force. Therefore, the input power 240 mW was used to separate this mixture. The second sample consisted of two different biological cells, purified human PBMCs and E. coli bacteria. PBMCs had an averaged diameter around 7.23 μm; while E. coli bacteria have an averaged volume similar to a sphere with a diameter of 1.1μm. The applied input power was 468 mW. The flow rates of the cell mixture and sheath flow were 0.5 μL/min and 4 μL/min, respectively. The separation efficiency of E. coli and PBMCs were 95.65% and 91.48%, respectively.In Reference , the sepAs in References ,10,11,12In this paper, The SSAW force with Rayleigh angle effect and its attenuation in liquid-loaded substrate, viscous drag force, hydrodynamic force, and diffusion force are analyzed. Also, the design analysis of SSAW microfluidics for the separation of particles is presented and discussed in details. The design analysis of SSAW microfluidics is depending on three factors. The first factor is the setup properties of the SSAW microfluidics such as: piezoelectric substrate material, input power, wavelength, length of IDTs, width of the main microchannel, widths of outlet collecting microchannels, working area of SAW, working time of SAW, and flow rate of fluid. The second factor is the properties of the fluid such as: density, compressibility, and viscosity. The third factor is the properties of the particles such as: density, compressibility, volume and radius.The design analysis of SSAW microfluidics is provided for selecting the piezoelectric substrate material, width of the main microchannel, working area of SAW, wavelength, minimum input power, and widths of outlet collecting microchannels required for the separation process. The minimum input power required to shift the maximum volume of the particles to the side wall of the main SSAW microchannel at the end of the working time of SSAW is calculated by simulation programs. The calculated minimum input power required for the separation process is determined with appropriated displacement contrast of the particles. Also, the simulations are provided for minimum power consumption, less complicated setup, and high collecting efficiency compared to other published papers ,10,11,12The simulation of the separation of blood components  based on the design analysis of SSAW microfluidics is presented. Also, the analysis of SSAW force with Rayleigh angle effect and its attenuation in liquid-loaded substrate, viscous drag force, hydrodynamic force, and diffusion force are presented and discussed.ϕ). The acoustic radiation force (SSAWF) is represented as following [The Surface acoustic waves (SAWs) are generated by two IDTs. These SAWs are overlapped and produced SSAW to generate the acoustic radiation force. Therefore, pressure node and antinode are generated. These lead the particles to migrate toward the pressure node or antinode depending on the sign of the acoustic contrast factor (ollowing :(3)FSSAWϕ can be expressed as following:p0, V, λ, ρp, ρm, βp, βm, k, and x are the acoustic pressure amplitude, the volume of the particle, the wavelength, the density of particle, the density of medium, the compressibility of particle, the compressibility of medium, the wave number, and the distance from the pressure node to the initial particle position, respectively. As shown in Equation (4), the acoustic contrast factor (ϕ) is determined by the density and compressibility of the particles and the medium.The acoustic contrast factor p0) can be calculated as following:P, Z and A are the AC signal power applied to IDT, the acoustic impedance of the piezoelectric substrate, and working area of SAW, respectively. The acoustic impedance of the piezoelectric substrate (Z) can be calculated as following:sρ is the density of the piezoelectric substrate. The power flow in SAW devices can be detected by high sensitive optical measurement [The acoustic pressure amplitude  determines whether the particle will move towards the pressure node or the pressure anti-node. If ϕ > 0, the particles will be moved to the pressure node. If ϕ < 0, the particles will be moved to the pressure antinode. The most biological particles in liquid medium are moved toward pressure node [The acoustic contrast factor ure node .vF) is given by Stoke’s law as following [η, v, and r are the fluid viscosity, the relative velocity between particles and medium, and the radius of the particle, respectively. The acoustic radiation force is dominant compared to the viscous drag force. Also, the viscous drag force cannot be neglected.The viscous drag force is the resistive force acting on particles in the opposite direction of acoustic radiation force. The viscous drag force is derived from Stoke’s law which describes the motion of sphere particles in a viscous fluid at a specific velocity. The viscous drag force (ollowing :(7)Fv=6πX1 to position X2 based on SSAW and viscous drag forces only (tSSAW) can be expressed as following [When the particles have constant velocity in the SSAW field, the acoustic and viscous forces are balanced. According to Equations (3), (4), and (7), the time required for the particle to migrate from position ollowing :(8)tSSAWSSAWd) based on the SSAW and viscous drag forces can be approximately expressed as following [wt is the working time of the acoustic radiation force on the particle.The displacement of particles (ollowing :(9)dSSAWRθ) is the refraction angle produced due to the changing in SAW propagation velocity in piezoelectric substrate and the liquid medium. The Rayleigh angle (Rθ) can be calculated by Snell’s law as following:mv is SAW velocity in the medium. When SAW meets the edge of the liquid, the energy will couple into the liquid at the Rayleigh angle [λ/2 or more. Therefore, the Rayleigh angle can not be neglected. As shown in SSAWF sin (Rθ).The Rayleigh angle  can be calculated as following [SAWl is the attenuation length of SAW in liquid-loaded substrate. The attenuation coefficient (α) is depending on the properties of liquid and piezoelectric material substrate; and the wavelength of SAW. The attenuation is increased exponentially with the attenuation coefficient.The attenuation in liquid-loaded substrate affects on the horizontal component of SSAW force. This attenuation is in ollowing :(12)FSSAollowing :(13)α=1lFSSAW) with Rayleigh angle (Rθ) effect and its attenuation in liquid-loaded substrate can be expressed from Equations (3) and (11)–(13) as following:FSSAW All is the acoustic radiation force with Rayleigh angle effect and its attenuation in liquid-loaded substrate.The acoustic radiation force  and pressure anti-nodes (maximum pressure amplitude) as shown in λ/2, without the attenuation effect, the locations of pressure nodes will be exactly on the both sides of the SSAW microchannel. On the other hand, with the attenuation effect, the locations of pressure nodes will be slightly moved toward the center of the SSAW microchannel. Therefore, if the SSAW microfluidics is designed to migrate the particles completely to the side wall of SSAW microchannel (pressure node), the particles will not move completely to the desired locations due to the effect of the attenuation of acoustic radiation force.Without the attenuation effect, the equilibrium force line will be at zero force line. Regarding to the attenuation of acoustic radiation force, the locations of pressure nodes will be affected. As shown in X1 to position X2 based on SSAW force with Rayleigh angle effect and its attenuation in liquid-loaded substrate, and viscous drag force (tSSAW All) as following:The Equation (8) can be modified to express the time required for the particle to migrate from position dSSAW All) based on SSAW force with Rayleigh angle effect and its attenuation in liquid-loaded substrate, and viscous drag force can be approximately expressed as following:The displacement of particle (fW) in rectangular microchannelsare explained in details in [fW) can be calculated using mass conservation law as following [iQ, sQ1, sQ2, oW, γ are the volumetric flow rate of the inlet microchannel, the volumetric flow rate of the side microchannels 1 and 2, the width of the main microchannel, and the velocity ratio of the average velocity of the focused stream to the average velocity of the stream in the outlet microchannel which assumed as a unity [Hydrodynamicd) from the center of the main microchannel based on the hydrodynamic force can be calculated according to Equation (18) as following:The hydrodynamic force is generated due to the flow of the core stream in the inlet microchannel among the flow of symmetric side microchannels. The core stream will be focused in the center of the main microchannel. The assumptions which used to determine the width of the focused stream  due to SSAW force with Rayleigh angle effect and its attenuation in liquid-loaded substrate, viscous drag force, hydrodynamic force, and diffusion force can be approximately expressed as following:The total displacements of the particles  compared to the others.The piezoelectric substrate material is important to generate the SAW. The piezoelectric substrate material is the main factor of the value of the horizontal component ratio of SSAW force due to the effect of Rayleigh angle as shown in Equation (11). The horizontal component ratios of SSAW force in distilled water for LiNbOThis proposed design analysis is provided to separate the particles types have different properties .λ) is inserted. Then, the properties of the setup of SSAW microfluidics device are calculated based on the mathematical models which described in the previous chapter as following: λ/5).The width of the main SSAW microchannel is about (2λ/20).The wall thickness of the main microchannel is about  at the end of the working area of SSAW force are calculated.R1 + 3R2). Where: R1 and R2 are the radiuses of the first the second particle types, respectively.The calculated difference between two types of particles is about  are accepted, the dimensions of output microchannels can be calculated. The separation is accepted under the following conditions:NOutputMicrochannels) can determined as following:n is the number of the particles types. The particle type which has the minimum effect of SSAW force will collected from the middle output microchannel. Two-symmetric output microchannels are designed to collect each other particles type.The number of output microchannels (λ) will incremented; then, the previous steps will be repeated.If the differences among the separated particles (∆Xs) are not accepted, the wavelength  is the dominant property affecting on the separation process. The values of radius, density, and compressibility of WBCs, RBCs, and platelets are shown in 3, 448 T·Pa−1, and 1 mPa·s, respectively. The simulation programs are built by MATLAB.In our simulation programs, the selection of minimum input power required for the separation process, piezoelectric substrate material, wavelength, width of the main microchannel, widths of outlet collecting microchannels, and working area of SAW are the main target in our design analysis of SSAW microfluidics for the separation of blood components. The constant values of the length of IDTs and working time of SAW are 9 mm and 0.9 s, respectively. The constant values of the ratio of the flow rates of input inlet microchannel to the symmetric side microchannels are used to provide 0.9 s working time of SAW. To simplify the simulation in this study; therefore, the distilled water is used as a fluid carrier of blood components and as sheath fluid. The density, compressibility, and viscosity of distilled water are 1000 kg/mIn the design analysis, the minimum input power required to shift the maximum volume of the particles to the side wall of the main SSAW microchannel at the end of the working time of SSAW is calculated and used to decrease the power consumption in SSAW microfluidics. The calculated minimum input power has relationships with the properties of particles, fluid, and other properties of SSAW microfluidics.2, and LiNbO3, respectively. The radius of particle is varying from 1 to 7.5 μm. The acoustic contrast factor is varying from 0.1 to 0.75. The required input power is inversely proportional to radius of particle and acoustic contrast factor.The relations of the minimum input power required to shift the particle to the side wall of the main SSAW microchannel at the end of the working time of SSAW, radius of the particle, and the acoustic contrast factor are shown in λ/5 to avoid the slight moving of the pressure nodes due to the attenuation effect of SAW. Therefore, the relation between the width of the main SSAW microchannel and the wavelength is shown in In our design analysis, the width of the main SSAW microchannel is selected as 2The relation between the minimum input power required to shift the WBCs (the maximum particle compared to RBCs and platelets) to the side wall of the main SSAW microchannel at the end of the working time of SAW and the wavelength is shown in The selection of the minimum input power required for separating the blood components WBCs, RBCs, and platelets is the main target in the design analysis. The shifting of the maximum particles (WBCs) to the side wall of the main SSAW microchannel at the end of the working time of SSAW is the reference to calculate the minimum input power. This minimum input power is applied to all blood components. Therefore, the displacement contrast or the difference of the displacement of particle from the center of the main SSAW microchannel (∆X) is essential when selecting the required input power. The relations between the difference of particle radius (∆R) (the reference particle is WBC) and the difference of the displacement of particle from the center of the main SSAW microchannel (∆X) at selected values of wavelengths are shown in The ∆R between WBCs and RBCs (5 μm–3.5 μm) is 1.5 μm, and the ∆R between WBCs and platelets (5 μm–1.5 μm) is 3.5 μm. At 300 μm wavelength, the displacement contrast (∆X) between WBCs and RBCs is approximately 31 μm, and the displacement contrast (∆X) between WBCs and platelets is approximately 51 μm. From these displacement contrasts, the calculated displacement contrast (∆X) between RBCs and platelets is approximately 20 μm. These displacement contrasts appropriate to optimize the separation process with minimum required input power. Therefore, the 300 μm wavelength is selected for the design analysis. As shown in Most previous SSAW-based devices as in ,10,11,12λ/5) and 25 μm, respectively. The lengths of the main SSAW microchannel and IDTs are 15 mm and 9 mm, respectively. The SAW wavelength (λ) is 300 μm. The SAW piezoelectric substrate material is LiNbO3. The flow rates in the input inlet microchannel and the two symmetric side microchannels are 0.2 μL/min and 0.8 μL/min, respectively. The mixture of blood components flows in the input inlet microchannel. The distilled water is used as a fluid. The working time of SSAW force is 0.9 s. The minimum required input power is 371 mW.The final SSAW microfluidic design for the separation of blood components  is based on the schematic shown in The simulation of the separation of blood components  based on the design analysis of SSAW microfluidics and the analysis of SSAW force with Rayleigh angle effect and its attenuation in liquid-loaded substrate, viscous drag force, hydrodynamic force, and diffusion force is shown in The simulation of the separation of blood components  based on the design analysis of SSAW microfluidics and the SSAW force only is shown in In Reference , the sepThese results of the separation of the blood components  demonstrate the design analysis of SSAW microfluidics and the analysis of SSAW force with Rayleigh angle effect and its attenuation in liquid-loaded substrate, viscous drag force, hydrodynamic force, and diffusion force. The analysis study is significant and powerful for the simulation of separation process especially for separation of biological particles.This paper demonstrated more accurate theoretical simulation of the separation of particles using SSAW microfluidic devices. The SSAW force with Rayleigh angle effect and its attenuation in liquid-loaded substrate, viscous drag force, hydrodynamic force, and diffusion force were analyzed. The design analysis of SSAW microfluidics for the separation of particles was presented and discussed in detail. The design analysis of SSAW microfluidics was depending on three factors. The first factor was the setup properties of SSAW microfluidics such as: piezoelectric substrate material, input power, wavelength, length of IDTs, width of the main microchannel, widths of outlet collecting microchannels, working area of SAW, working time of SAW, and flow rate of fluid. The second factor was the properties of the fluid such as: density, compressibility, and viscosity. The third factor was the properties of the particles such as: density, compressibility, volume, and radius.The design analysis of SSAW microfluidics provided the selection of the piezoelectric substrate material, width of the main microchannel, working area of SAW, wavelength, minimum input power, and widths of outlet collecting microchannels required for the separation process. The minimum input power required to shift the maximum volume of the particles to the side wall of the main SSAW microchannel at the end of the working time of SSAW was calculated by the simulation programs and used to decrease the power consumption.λ/5 to avoid the slightly moving of the pressure nodes due to the attenuation effect of SAW. The design analysis of SSAW microfluidics achieved the minimum input power required for the separation process with appropriated displacement contrast of the particles. The relations between the difference of particle radius (∆R) and the difference of the displacement of particle from the center of the main SSAW microchannel (∆X) at selected values of wavelengths were presented.The relations of the minimum input power required to shift the particle to the side wall of the main SSAW microchannel at the end of the working time of SSAW, radius of the particle, and the acoustic contrast factor were presented. The width of the main SSAW microchannel was selected as 2λ/5) and 25 μm, respectively. The lengths of the main SSAW microchannel and IDTs were 15 mm and 9 mm, respectively. The SAW wavelength (λ) was 300 μm. The SAW piezoelectric substrate material was LiNbO3. The flow rates in the input inlet microchannel and the two symmetric side microchannels were 0.2 μL/min and 0.8 μL/min, respectively. The mixture of blood components will flow in the input inlet microchannel. The distilled water was used as a fluid. The working time of SSAW force was about 0.9 s. The minimum input power required for the separation of blood components was 371 mW. All simulation programs were built by MATLAB.The final design of SSAW microfluidics for the separation of blood components  was analyzed. The width and height of the main SSAW microchannel were 120  based on the design analysis of SSAW microfluidics was presented. The analysis of SSAW force with Rayleigh angle effect and its attenuation in liquid-loaded substrate, viscous drag force, hydrodynamic force, and diffusion force was discussed and analyzed. The displacement of WBCs, RBCs, and platelets at the end of the working time of SSAW force due to the SSAW force with Rayleigh angle effect and its attenuation in liquid-loaded substrate, viscous drag force, hydrodynamic force, and diffusion force were 60, 29, and 9 μm, respectively. The separated particles were simply collected by five outlets microfluidics. The widths of the five outlets microfluidics were determined. The width of middle outlet microchannel for platelets collection was about 30 μm. The width of two mid-lateral outlet microchannels for RBCs collection was about 20 μm. The width of two lateral outlet microchannels for WBCs collection was about 25 μm.The simulation of the separation of blood components  based on the design analysis of SSAW microfluidics and the SSAW force only was presented. Under the same simulation conditions, this simulation presented obviously the importance of other forces and effects. The SSAW force only was not enough to provide accurate simulations.This study provided accurate design and simulations for the separation process. The results of these simulations achieved minimum power consumption, less complicated setup, and high collecting efficiency compared to other published papers. This analysis study was significant and powerful for the simulation of the separation process especially for the separation of biological particles.


This mode of action, facilitating cooperation between proteases on both sides of the thylakoid membranes, should be applicable to the degradation of other integral thylakoid membrane proteins as well.Degradation of the D1 protein of photosystem II (PSII) reaction center is a pre-requisite for the repair cycle from photoinhibition. Two types of thylakoid proteases, FtsH and Deg, have been demonstrated to participate in this process. However, the location of the proteolytic sites of the lumenal Deg1 protease within its internal sphere raised the question whether the lumenal-exposed regions of D1 are indeed long enough to reach these sites. Implanting these regions into the stable GFP rendered it sensitive to the presence of Deg1  Their concerted action provides the chloroplast with NADPH and ATP, the reducing power and energy source, respectively, necessary for CO2 fixation. Most of the subunits of these multi-protein complexes are integral membrane proteins. Proper operation of this machinery under changing environmental conditions requires turnover of different subunits of these complexes at different rates. Thus, proteolytic degradation of specific chloroplast proteins is inherent to the capability of plants to maintain photosynthesis under different physiological conditions.The thylakoid membrane houses the heart of the photosynthetic machinery, composed of four major complexes: photosystem I and photosystem II (PSII), capable of harvesting light energy and converting it into a chemical one, the cytochrome 2). This degradation of the D1 protein is a prerequisite for the incorporation of a newly synthesized D1 into the damaged complex and reactivation of PSII. As other thylakoid membrane proteins are also prone to irreversible oxidation by reactive oxygen species, although at a lower rate compared to D1, they are also subjected to proteolytic degradation. One of the preventive mechanisms to minimize the risk of oxidation damage induced by high light intensities, and hence of photoinhibition, involves adjustment of the size of the photosynthetic antenna, by degradation of chlorophyll binding proteins, which leads to a decrease in the excitation pressure. Another example of selective proteolytic degradation of a photosynthetic complex is that of the cytochrome b6-f complex, during the response of the chloroplast to nutrient deficiency3.The most studied proteolytic event in the thylakoid membrane is that of the D1 protein of PSII, in the context of PSII repair from oxidative damage incurred by exposure to light that may lead to a phenomenon known as photoinhibition  are located within a cage-like structure9  raised the question whether hydrophilic segments of the D1 protein, extruding into the lumen .The dimensions of these pores and their distance from the proteolytic active sites  connecting the first and second trans-membrane helices, the CD loop (Gln165 – Leu193) connecting the third and fourth helices, and the C-terminus of D1 (Asn296 – Ala344) see Fig. . Both loE. coli. These results demonstrate that the unfolded loops of D1 protruding out of the tightly folded cylinder-like structure of the GFP, as well as the extension at its C-terminus, are long enough to penetrate through the side pores of Deg1 and reach its active sites, which, as mentioned above, are 30–40 Å away. Since we could not detect any distinct fragments of the substrates even at shorter incubation times, we suggest that once an initial cleavage has occurred, the substrate GFP unfolds and is immediately degraded into very short peptides or free amino acids, consistent with the processive nature of Deg proteases7. These results support the notion that the segments of the D1 protein that are exposed to the lumen can be targeted and cleaved by the Deg1 protease.The reaction mixture of the proteolytic assay contained 50 pmol of the substrates and 10-fold less of the different proteases. After incubation, the reaction mixtures were resolved by SDS-PAGE and the remaining substrate bands were quantified by scanning densitometry. Representative gels are shown at the top of Fig. In the fully assembled PSII complex, the segments of the D1 protein that are oriented towards the lumen are normally shielded by the oxygen-evolving complex (OEC) that is peripherally attached to the PSII core complex. In this conformation, these segments are protected from proteolysis by Deg1 or any other lumenal protease. This implies that a prerequisite for their cleavage by Deg1 is the detachment of OEC from the holo-PSII complex. Thus, proteolytic attack on the lumenal side of D1 is regulated at two levels: at the substrate level – partial disassembly of PSII, likely induced by oxidative damage, that exposes hydrophilic segments of D1 to the lumen; at the protease level – activation of Deg1 by its hexamerization, that is induced by acidification of the lumen. As both oxidative damage and lumen acidification result from the exposure of the photosynthetic machinery to light, the two levels of regulation are synchronized.11, it is not the only thylakoid membrane protein that undergoes proteolytic degradation. All integral membrane proteins are anchored to the membrane by at least one trans-membrane helix, and most of them contain hydrophilic segments protruding to the lumen. We propose that, similar to the D1 protein, these segments might be targets of Deg1 protease, facilitating complete degradation of thylakoid membrane proteins, in cooperation with proteases on the stromal side of the thylakoid membrane, such as the FtsH complex.Although the D1 protein of PSII is one of the fastest turning over proteins in the chloroplastArabidopsis thaliana (AtCg00020) . The AB loop (Pro56 – Gly110) and the CD loop (Gln165 – Leu193) were inserted between Pro211 and Asn212 of GFP, and the C-terminus of D1 (Asn296 – Ala344) was fused downstream of that of GFP. All constructs contained 6xHis tag at their C-terminus. These constructs, as well as those encoding His-tagged Deg1 from Arabidopsis thaliana10 and DegP and DegQ from E. coli7, were expressed in bacterial cells, and affinity purified on Ni-NTA columns, as previously described10. Proteolytic degradation assays were conducted essentially as in10. Briefly, 50 pmol of substrates and 5 pmol of proteases were mixed in 50 mM MES, pH 6.0, in a total volume of 20 μl, and incubated at 37 °C for 2 hrs. The reactions were terminated by adding SDS-solubilization buffer and resolved by SDS-PAGE, and the remaining substrate bands were quantified by scanning densitometry.DNA sequences encoding the lumenal-exposed regions of the D1 protein from see Fig.  were fus


Despite the multi-systemic effects of influenza A/H1N1 virus, the occurrence of hepatic injury during the natural course of the infection remains a matter of debate. We performed a review of the published clinical studies which assess the above mentioned relationship, reviewing the studies published in PubMed database (English literature), using the key words “H1N1”, “influenza A” and “liver”. We excluded case reports and clinical studies that referred to pediatric and transplanted patients, pregnants and patients with known history of chronic liver diseases. From a total of 96 results, a total of 78 papers met one or more of the exclusion criteria set. Evaluating the remaining 18 published papers, 14 more were excluded as they did not provide any sufficient data, relevant to the subject of our review. Although the analysis of the remaining studies revealed the existence of conflicting results concerning the exact degree and the potential mechanisms of liver injury in H1N1 positive patients, it can be assumed that influenza A/H1N1 virus is -or at least could be- a hepatotropic virus. The outbreak of the influenza A/H1N1 virus in 2009 has undoubtedly been a major challenge for the health systems worldwide. As a result of its rapid spread and clinical severity, it was officially recognized by the WHO on the 11th of June 2009 as the first pandemic of the 21st century -6. When Focusing on the effect of this viral infection with respect to the liver function, there are relatively limited studies in the international literature that attempt to evaluate the latter, with the research topics spanning from retrospective reports of the alterations in liver biochemistry during the course of influenza A/H1N1 infection to pathology studies of the liver in patients who succumbed due to multiple organ dysfunction system, triggered by the virus. Without a doubt, in terms of laboratory essays, the values of the alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyltransferase (GGT) stand as the predominant screening examinations of the functional status of the liver . ConsequWe reviewed the studies published in PubMed database (English literature), using the key words “H1N1”, “influenza A” and “liver”. We excluded case reports and clinical studies that referred to pediatric and transplanted patients, as well as pregnants and patients with known history of chronic liver diseases . The literature search was performed by three of the co-authors, working on an independent basis.We identified 4 (four) retrospective clinical studies, in which were documented the admission values of the commonly used liver enzymes.One of them, performed by Chen Yingying , evaluatRegarding the remaining three retrospective studies which assess the existence of a degree of liver injury as a result of the infection by the Influenza A/H1N1 virus, all of them attempt to compare the different extent of liver dysfunction between the seasonal influenza and influenza A/H1N1 during the pandemic of 2009. One of these three, performed by Papic et al  aimed spSpecifically, Papic et al , compariOn the contrary, Zarogoulidis et al , who alsThe study by Wang et al  appears Considering all the results of the above mentioned studies, it becomes evident that one of the key reasons the influenza A/H1N1 pandemic tested the healthcare systems worldwide was the capability of the virus to impair the function of all vital organs and trigger the cascade of systematic inflammation, even in young patients . What is


The aim of this work is to study the influence of the admixture of superplasticizers on the physical properties of cement paste to know the initial and final time, water content and mechanical performance of concrete.In this work we have incorporated an Advanced Superplasticité for Prolonged Slump Retention (ASPPSR402) in the formulation matrix of concrete at different percentages ranging from 0.5% to 5% by weight of cement with a step of 0.5%, while partially substituting the mixing water by the last one to reduce the amount of water used.The obtained results by different prospected formulations show that the admixture of ASPPSR402 in formulations matrix significantly reduces the (Water/Cement “W/C”) ratio. Subsequently, we observed that the initial and final time increases in function of the percentage of ASPPSR402. Similarly, the compressive strength at the young age, median age and long-term has been improved. Specifications TableValue of the data●Evaluation of the W/C ration, setting time and the compressive strength of Portland cement  compared to the cement  based on the various volume of advanced admixtures of superplasticizer for prolonged slump retention.●Estimation of the evolution of the ratio (W/C) and the initial and final time of cement paste as a function of the various percentages of the ASPPSR402 and the amount of water reduced according to the various percentages of ASPPSR402.●Estimation the rate of change compressive strength of ordinary Portland cement as a function of curing time.●Optimization of the curing time to achieve a particular rate of change of compressive strength of using the admixture of ASPPSR402.1The data of the different formulations in a fresh and hardened state presented here are from eleven different cement pastes, mortar or concrete samples fabricated to compare cement pastes, mortar or concrete based of ASPPSR402 with Portland cement based concrete.2To achieve the objective of our study, we made a mortar of reference without additions and ten samples with the ASPPSR402 whose compositions are inspired by that of the normal mortar defined by EN196-1, with a quantity of water adjusted to obtain a paste with a standard consistency. The procedures followed for the preparation of the pastes cement has been done in maintaining fixed consistency standardized of all the formulations produced according to the specification of the standard (EN 196-3 + A1) 3 according to the specification of the standard (NF EN 196-1) To complete our work, we also studied the influence of the ASPPSR402 on the mechanical performance of mortar or concrete by using the compressive strength. The measures of the compressive strength are on standard prismatic standard (4 × 4 × 16) cmThe compositions of the different formulations in a fresh and hardened state are recorded in the , Table 233.1For evaluating the influence the Admixtures of advanced superplasticiser for prolonged slump retention "ASPPSR402", on physical properties of fresh cement paste and mechanical performance  of mortars and /or concretes in the hardened State. We have preceded our work by the characterizing of materials used, to understand the phenomena which occur at the moment of mixing and hardening of concrete.3.1.1The type of cement used in this work is (CMI/42.5) from the plant of Amran in Yemen. The chemical and mineralogical compositions of clinker, gypsum and cement determined by XRF are presented in the 3.1.1.1The physical properties of clinker and cement are gathered at the 3.1.2To prepare our mortar, we used a standard sand conferring to the standard EN 196-1 3.1.3The Advanced superplasticizer for Prolonged Slump Retention "ASPPSR402"), are polymers in liquid form, prepared especially for the cement industry and concrete. They are a basis of salts of sodium or calcium of poly-naphthalene sulfone , salt ofThe ASPPSR402 used in the formulation of mortars and/or concrete of high performance, delivered by the company CONMIX Ltd in Sharjah, United Arab Emirates. They are incorporated during the mixing of the concrete in equal a dose raining from 0.5% to 5% by weight of cement with a step of 0.5% to improve the physical and the mechanical properties of fresh or hardened State.The physical properties of ASPPSR402 are collected at the 3.1.4To waste our mixture, we used tap water (wells). The physical properties of mixing water are assembled at the 3.23.2.1The 3.2.2The 3.3The MC' at different percentages of ASPPSR402.The


Urolithiasis is a significant social and financial problem. According to contemporary literature data, 1-5% of the global population suffers from urolithiasis. The prevalence of this disease is about 10% of the population of the Republic of Moldova. Scientific and practical researches in the field of urology, and especially those devoted to renal lithiasis, focus on the diagnosis and treatment process, giving little importance to the cause of formation, metabolic disturbances, and especially to prophylaxis and metaphylaxis of the disease recurrence. However, the impact of this disease may be diminished by specialized or general metaphylaxis treatment. The article presents results of the analysis of different methods of metaphylaxis of recurrent urolithiasis. The implementation of metaphylaxis measures significantly reduces the rate and risk of recurrence in patients with recurrent urolithiasis. Specialized metaphylaxis treatment reduces the risk of lithiasis recurrence 5 times and general metaphylaxis - 2 times compared to the lack of metaphylaxis, which requires this treatment to prevent the recurrence of urolithiasis. Over the last decades, a progressive increase in the incidence of urolithiasis was recorded on the entire earth globe . According to contemporary literature data, 1-5% of the global population suffers from urolithiasis, but the incidence of this pathology varies depending on geographical area. The risk of urolithiasis in adults is higher in the western hemisphere  compared to the eastern hemisphere (1-5%), although the maximum incidence was registered in some Asian countries such as Saudi Arabia (20.1%) . The prevalence of this disease is about 10% of the population from the Republic of Moldova . The incidence of urolithiasis also depends on both racial distribution and socio-economic status of the surveyed population.Urolithiasis currently occupies one of the leading places in the structure of urological diseases being qualified as a major medical-demographic problem . There is also an increase of urolithiasis incidence and prevalence in the Republic of Moldova, which from 2005 has become the first in the structure of diseases in urological clinics, which places secondly some pathology as inflammations and prostate adenoma [10]. In male urolithiasis, males are affected more often, at a rate of 52-60%. Male/ female ratio is 1:3 or 1:2. Some authors, however, found much higher figures due to excessive working regimen, food abuse, urethro-prostatic disease and other factors that determine an increased prevalence of lithiasis in men .An annual increase of urolithiasis incidence in economically well-developed countries is 2-2.5% , but in the Russian Federation, this index ranges from 1% to 3% [1-3] in clinical practice. These methods occupy one of the first places in the curative process.In 1980s and 90s of the last century, the basic methods of treatment of urolithiasis were conservative methods and surgical techniques. At present, significant progress is, along with extracorporeal shock wave lithotripsy (ESWL), the implementation of modern endourological techniques . However, a detailed review of literature demonstrates that scientific-practical research in urology, and especially those devoted to renal lithiasis, focuses on the diagnosis and treatment process, giving little importance to the cause of formation, metabolic disturbances and in particular to prophylaxis and metaphylaxis of the disease recurrences . After a thorough analysis of specialty literature on prophylaxis and metaphylaxis of urolithiasis recently published in PubMed electronic database, there has been an increase in the number of articles published in this field over the last 7 years .Until now, great experience has been gained in the treatment of urolithiasis. The success of treatment is largely determined by the chosen tactics and curative method. The methods applied and their combination in multimodal treatment of pathology, result in total stone removal (stone-free) [16].In their study, Meneses JA et al. (2012) showed that in patients with recurrent urolithiasis pathology, who did not perform metaphylaxis measurements, both the occurrence of chronic renal disease and the recurrence rate were higher compared to patients who underwent metaphylaxis. The recurrence of disease leads to partial or total loss of renal functional capacity, which sometimes progresses to chronic renal failure and invalidity of patients with decreased ability to work and quality of life . Until now, there has been no single opinion on the type and volume of metaphylaxis medical manipulation that would be optimal to be performed after the removal of urinary concretions.The diversity of the causes of calculi occurrence but also of clinical forms of urolithiasis, chemical composition, localization, present urinary infection, complicate the prophylaxis and metaphylaxis of this pathology, which needs to be viewed individually in each case as far as possible [The purpose of the research was to evaluate the efficacy of metaphylaxis in patients with recurrent urolithiasis. The trial was conducted in the clinic of Urology and Surgical Nephrology, “Nicolae Testemitanu” State University of Medicine and Pharmacy, Republican Clinical Hospital during the years 2010 and 2014 based on clinical and laboratory data from 160 patients with recurrent kidney lithiasis, who were treated in inpatient and outpatient departments.The inclusion criteria were the following: patients with recurrent renal lithiasis who signed the study participation consent. The exclusion criteria were the following: patients who refused to sign the study participation agreement or were diagnosed with Chronic Renal Disease K-DOQI degree IV-V and/ or other concomitant serious illnesses.The study was approved by the Research Ethics Committee of “Nicolae Testemitanu” SUMPh, Chisinau, Republic of Moldova.Patients were randomly assigned to research groups to determine the efficacy of metaphylaxis. Group I included 58 patients of total number of investigated patients who underwent a complex metabolic evaluation, after which, patients were prescribed individual metaphylaxis treatment recommendations. Group II included 52 patients with recurrent calculi, who were recommended after inpatient treatment and general measures against recurrence of urolithiasis. Group III included 50 patients with recurrent urolithiasis, who were treated in the Urology SCR Department, were questioned 3 years after the treatment, but failed to fulfill the disease prevention recommendations.Fig. 1.The study design (CONSORT-chart) is shown in The comparative efficacy of the treatments was assessed by the following tests: Pearson’s chi-square, Hazard, Kaplan-Meier, RR - relative risk and NNT (Number Needed to Treat) - the number of patients necessary to treat to avoid an event. The data were processed by using statistical software package SPSS 21.Table 1).All 160 (100%) patients completed the study. According to age distribution, 10.6% of the cases were between 18 and 30 years old, 65.6% - 31 and 60 years old and 23.8% - over 60 years old  of the cases, which confirmed the social impact of the pathology affecting the working life period.Analyzing Table 2. The distribution of patients on research groups is presented in Table 2 we noted that the distribution of patients by groups, according to age and method of treatment previously applied, which was homogeneous, the groups were comparable and the statistical significant differences between study groups were not found (p > 0.05).Analyzing the data in Table 3).During the follow-up (36 months) of patients with recurrent urolithiasis from those three groups (160 patients) included in the study, recurrence of urolithiasis was detected in 42 patients (from the whole cohort) (Distribution of urolithiasis recurrence rates between the investigated groups was significantly non-homogeneous (χ2 (2) = 14.2, p = 0.0008).Fig. 2.Hazard rate (HR) function of urolithiasis recurrence appearance for the three studied groups is shown in Fig. 2, the risk of recurrence increased concurrently with the time interval increase from the removal of recurrent calculus. Therefore, the risk of recurrence appearance in patients with recurrent urolithiasis one year after the calculus removal was of about 8%, in two years – of about 22%, and in three years of about 30%. As shown in The lowest rate of urolithiasis recurrence occurred in Group I patients, accounting for 12.1% (7 out of 58 patients). In 13 patients (25.0%) in Group II (n = 52) the recurrence of urolithiasis was determined. The highest rate of recurrence occurred in Group III patients and accounted for 44.0% (22 out of 50 patients).Table 4) showed that the risk of urolithiasis recurrence in Group I patients was 4.55 times lower compared to Group III, with a statistical significance (p = 0.0001).The relative risk of urolithiasis recurrence in patients who, after the metabolic analysis, followed the specialized metaphylaxis recommendations (Group I) was RR=0.22 =13.9; p=0.0001). The data obtained (Table 4), it was determined that the risk of recurrence of urolithiasis in this group of patients (Group II) was 2.02 times lower compared to the risk in Group III patients, with a statistical significance (p = 0.044). Although insignificant, the relative risk of recurrence development was higher in group II patients compared to patients who met specialized metaphylaxis recommendations, which outlined the anti-recurrent efficacy of these measures.The relative risk of recurrence of urolithiasis in patients who met metaphylaxis general recommendations (Group II) was RR = 0.495  = 4.08; p = 0.044). Based on the obtained results .By using Kaplan-Meier curves, the time to recurrence of urinary calculi in the investigated and control groups was compared, determining the differences concerning the metaphylaxis treatment applied methods (Table 5).The differences between “time to recurrence” in the investigated groups are statistically significant, as evidenced by log-rank test (Table 6).While comparing pairs between specialized metaphysics (group I) and those with general metaphylaxis (group II), it was determined that the differences in the recurrence-free interval of urolithiasis were insignificant and the meaningful tests performed between these groups revealed p value > 0,05  to prevent the recurrence of urolithiasis, was calculated as reciprocal of absolute risk reduction of developing a recurrence of urinary lithiasis.Thus, when calculating the absolute risk of urolithiasis recurrence in the control group for 3 years, it was determined that it represented 44%. At the same time, the absolute risk value in the group of patients benefiting from specialized metaphylaxis treatment was 12.07%. Therefore, the absolute risk reduction, defined as the absolute difference between urolithiasis recurrence rate in patients who underwent a specialized metaphylaxis treatment and a control group event rate, was 31.93%, with a confidence interval (CI 95%) within the limit of 15.82% to 48.04%. When calculating the required treatment number, its value, equal to 4, was obtained, which meant that one of four patients benefitted from a specialized metaphylaxis treatment, fact expressed in the absence of the recurrence of urinary lithiasis, compared to patients in the control group. The confidence interval (CI 95%) for the required number to be treated was 2.1-6.3.In the group of patients who underwent a general metaphylaxis treatment, 25% of them developed a recurrence of urolithiasis, this percentage indicating the absolute risk value. Thus, compared to the control group, general metaphylaxis treatment reduced the absolute risk of developing recurrences of urinary lithiasis by 19%, the confidence interval (CI 95%) for this index being from 0.89% to 37.7%. The NNT index for this type of treatment was 6, with a confidence interval (CI 95%) from 2.7 to 111.8. In order to prevent the development of recurrence of urolithiasis, it was necessary that 6 patients underwent a general metaphylaxis treatment.The recurrence rate of recurrent urolithiasis was frequent and increases with time more and more after the removal of renal calculus, and the risk of recurrence in patients with recurrent urolithiasis one year after the calculus removal was of around 8%, after two years – of about 22%, and after three years of approximately 30%. The implementation of metaphylaxis measures reduced the rate and risk of recurrent urolithiasis. Specialized metaphylaxis treatment reduced risk 5 times, and generally 2 times compared to the lack of metaphylaxis, which required the need for this treatment to prevent recurrences.