This study investigates the influence of design, relative density (RD), and carbon fiber (CF) incorporation parameters on mechanical characteristics, including compressive modulus (E), strength, and specific energy absorption (SEA) of triply periodic minimum surface (TPMS) lattice structures. The TPMS lattices were 3D-printed by fused filament fabrication (FFF) using polylactic acid (PLA) and carbon fiber-reinforced PLA(CFRPLA). The mechanical properties of the TPMS lattice structures were evaluated under uniaxial compression testing based on the design of experiments (DOE) approach, namely, full factorial design. Prediction modeling was conducted and compared using mathematical and intelligent modeling, namely, adaptive neuro-fuzzy inference systems (ANFIS). ANFIS modeling allowed the 3D printing imperfections  to be taken into account by considering the actual RDs instead of the designed ones, as in the case of mathematical modeling. In this regard, this was the first time the ANFIS modeling utilized the actual RDs. The desirability approach was applied for multi-objective optimization. The mechanical properties were found to be significantly influenced by cell type, cell size, CF incorporation, and RD, as well as their combination. The findings demonstrated a variation in the E (0.144 GPa to 0.549 GPa), compressive strength (4.583 MPa to 15.768 MPa), and SEA (3.759 J/g to 15.591 J/g) due to the effect of the studied variables. The ANFIS models outperformed mathematical models in predicting all mechanical characteristics, including E, strength, and SEA. For instance, the maximum absolute percent deviation was 7.61% for ANFIS prediction, while it was 21.11% for mathematical prediction. The accuracy of mathematical predictions is highly influenced by the degree of RD deviation: a higher deviation in RD indicates a lower accuracy of predictions. The findings of this study provide a prior prediction of the mechanical behavior of PLA and CFRPLA TPMS structures, as well as a better understanding of their potential and limitations.  Lattice structures are attracting attention in a wide range of industries, including aerospace, automotive, medical, and heat management, where lightweight and multifunctionality are required. Properties like thermal insulation, acoustic absorption, mechanical vibration damping, high stiffness-to-volume fraction ratio, and energy absorption are required within these structures . They enIn comparison to bulk materials, lattice structures have a high number of design variables, which increases their complexity and limits their ability to be fabricated using conventional manufacturing processes. However, the advent of additive manufacturing (AM) provides opportunities to fabricate such complex structures, e.g., lattice structures, and opens doors for creating and exploring more designs. The fundamental advantage of the lattice structures is that materials can be placed only where they are needed for a particular application . Thus, tThe most common lattice structures’ unit cells are strut-based and surface-based, e.g., triply periodic minimal surfaces (TPMS) ,12. StruResearchers are becoming increasingly interested in the design and 3D printing of TPMS structures in an attempt to enhance their performance. Spear and Palazotto  investigRegarding the incorporation of reinforcements as a strategy for enhancing lattices’ performance, very limited work has been reported particularly in TPMS structures. Zarei Zarei et al.  exploredIt is critical to understand the single and combined effects of various variables, such as material characteristics, relative density, and design parameters on the performance of the FDM TPMS structures. Studying the single and combined effects of various variables needs to be conducted based on a design of experiments (DOE) scheme. This allows statistical exploration of the single and combined effects of factors and further generalization of the lattice’s performance using model-based analysis. Developing models based on factorial design could be a beneficial method for designing customized lattice structures . To the It is worth mentioning that most of the reported studies ignored the influence of RD when statistically investigating the influence of the design parameters on mechanical characteristics. For instance, the reports that studied the effect of design parameters, such as strut/wall thickness, cell type, and cell size directly induced RD variations in their results. In other words, it can be said that the results were affected by RD in an uncontrolled manner. This is because wall thickness, cell type, and cell size control the RD, and any combination of them will lead to a particular RD. For example,  performeThis study attempts to examine the influence of material composition, geometry (cell type and size), and RD variables on the FDM-printed TPMS lattice structures using the DOE approach. In this regard, three TPMS cell types, namely Diamond, Gyroid, and Primitive, with various cell sizes (8 mm and 12 mm), CF incorporation (0% and 15%), and RDs (30% and 44%) were considered. The analysis was conducted based on a full factorial design. The actual relative densities of the printed samples were measured to ensure no significant difference between the designed and printed RDs. Uniaxial compression testing was used to evaluate the mechanical properties of 3D-printed samples, including compression modulus, strength, and SEA. Moreover, ANFIS modeling was also employed for predicting the performance of the TPMS structures, and the results were compared with the mathematical models developed based on the DOE approach. By using the ANFIS modeling predictions, we could accurately predict mechanical characteristics considering the inherent imperfections in 3D printing, such as an RD variation, which cannot be avoided. Due to the freedom in the ANFIS modeling approach, the actual RDs were used instead of the intended (designed) RDs, leading to improved prediction accuracy. Finally, the best parameter settings for maximizing the TPMS lattices’ performance were determined using multi-objective optimization through the desirability function. The following section discusses the materials and methods used in this study. The results are presented in 3  pure Polylactic acid (PLA) and (2) carbon fiber-reinforced PLA (CFRPLA) in which 15% high-modulus short carbon fibers CF were incorporated into the PLA matrix. The matrix of both materials is Natureworks 4043D PLA biopolymer grade. The materials (filaments) were provided by 3DXTech, USA, with a diameter of 1.75 mm, and used as received. For the pure PLA filament, the mechanical properties are 1.24 g/cmModulus) , while fModulus) .l) of all cell topologies was designed in a cubic unit cell with 8 mm and 12 mm edge lengths. The reason behind these particular lengths is to fit the whole lattice dimensions. In the current research, three different TPMS cell topologies, including Gyroid (G), Diamond (D), and Primitive (P) were considered. Within the context of this study, the terms “cell topology” and “cell type” are used interchangeably. lattice/Voverall), is defined as the lattice structure volume (Vlattice) divided by the overall structure volume  [t) to the cell size (t/l) [t/l ratio because of the differences in surface areas. The t values were determined based on the CAD predictions. t parameter for each cell topology at the designed RD and cell lengths.Relative density (RD) is one of the most factors that influence a lattice’s performance. RD, also termed as volume fraction  . This stze (t/l) . It shou3, see The lattice structures were designed and printed in sizes of 24 × 24 × 48 mmp-Values less than 0.05 imply that model terms (main factors and interactions) are statistically significant. Mathematical relationships between the investigated parameters and each of the output responses were developed. The developed mathematical models were further considered for optimization of the considered parameters. The best settings of the considered parameters were optimized through the desirability approach to simultaneously (multi-objective optimization) achieve the maximum E, compressive strength, and SEA. Design-Expert 13 software was used to systematically analyze the influence of the investigated parameters, develop mathematical (prediction) models, and conduct multi-objective optimization.The influence of the material composition (CF incorporation), cell topology, cell size, and relative density on the mechanical properties, including compression modulus (E) and strength, and specific energy absorption (SEA), was evaluated by the DOE approach. An open-source FDM machine, Prusa FDM printer , equipped with a 0.4 mm nozzle, was used to additively manufacture the lattice structures. The STL files for the TPMS lattice structures were imported into slicer software (PrusaSlicer 2.4.2) in order to slice and generate “GCODE” files. lattice) was then determined from the obtained density. Then, the ratio between , which represent the RD, was calculated. The overall volume  was calculated from the actual sample’ dimensions. Measurements were conducted on Shimadzu Analytical Balance  with a readability of 0.01 mg and a universal specific gravity kit  as in [The printed samples’ actual dimensions  were determined by a Lab Profile Projector VOM-2515), as shown in 15, as shThe influence of the considered parameters on the mechanical characteristics of the TPMS structures was conducted under a uniaxial compression test. The compression tests were carried out following the ASTM D695-15 standard. Compression tests were performed along the build direction at a constant crosshead speed of 1.6 mm/min up to 60% strain on a Zwick Z100 testing machine equipped with a 100 KN load cell. During the tests, forces and displacements were recorded using testXpert II software. peak) was considered as the peak strength, the maximum stress-value of the first peak  in the stress-strain curve [Mechanical characteristics, including compressive modulus (E), compressive strength, and SEA, were gathered from the force-displacement curves. First, engineering compressive stress was used and calculated by dividing the recorded forces (F) by the measured original cross-sectional area. The strain was computed by dividing the recorded displacements (δ) during the tests by the original length of the sample. Then, compression modulus and strength were determined. Compression modulus was calculated by the slope of the tangent line at the linear portion of the stress-strain curves by testXpert II software. Compression strength  is a useful indicator for measuring a structure’s capability to absorb energy per unit weight. The SEA of a lattice structure is represented by the area under the force-displacement curve divided by the structure’s weight. The SEA was calculated as in Equation (1) . The areANFIS is a hybrid neuro-fuzzy method for modeling complex systems. It integrates the best learning abilities of the artificial neural network (ANN) and inference capabilities of the fuzzy inference system (FIS) ,37. ANFILayer 1: input membership functions (MFs). In this layer, also called the fuzzification layer, the fuzzy membership value of each input, i.e., Layer 2: fuzzy rules. The firing strength .Layer 5: output layer. This layer has only one node to calculate the system output, as in Equation (5).The ANFIS modeling was used for predicting the performance of the TPMS structures in terms of E, σpeak, and SEA. Using the ANFIS modeling predictions, we could accurately predict mechanical characteristics considering imperfections in 3D printing, such as an RD variation. This way, the actual RDs were used instead of the intended RDs, leading to improved prediction accuracy. peak, and SEA of the 24 runs according to the full factorial DOE. The variabilty in the three repeated measured results is indicated by the standard deviation (SD). The results, including E, σpeak, and SEA, were derived from the force-displacement curves. Typical stress-strain curves under the uniaxial compression test of the 24 runs listed in Deformation mechanisms are graphically illustrated in peak, and SEA are 0.995, 0.999, and 0.97, respectively, which indicates an excellent representation of the variability of the data by the model terms. p-values less than 0.05 indicate that model terms are significant. From the ANOVA table , followed by cell type (23.87%). peak, whereas the Primitive-based cell type structures show the lowest. Compressive strength is proportionally influenced by RD; increasing RD results in an increase in σpeak  but decreased at low RD (30%) as the cell size increased.Similarly, the reduced ANOVA table for the σin σpeak . RegardiThe reduced ANOVA table for SEA is presented in peak, and SEA, and the investigated variables were developed based on the reduced models in the ANOVA analysis, e.g., Mathematical relationships between the responses, including E, σpeak, and SEA. An advantage of this type of modeling is that it eliminates the requirement to strictly adhere to predetermined settings for any variable in the DOE, where any change in that setting will most likely impact the results. For instance, the ANOVA analysis results , the accuracy of the developed ANFIS models was evaluated using the root mean square error (RMSE), which was calculated using Equation (6).In this study, the ANFIS models were developed during the training phase based on the full factorial results (as training data) presented in peak response is provided in Furthermore, for each TPMS performance, RMSE was used during the training phase for tuning and selecting the fuzzy inference parameters to minimize the RMSE. The fuzzy inference parameters and their settings that resulted in the lowest RMSE are presented in peak, and SEA based on the mathematical and ANFIS models are shown alongside the experimental results. peak, and SEA. Similarly, peak, and SEA compared with mathematical results. For instance, the peak is 0.977, which is significantly higher than that of mathematical prediction, which is 0.763. In peak), while a deviation of 21.11%  was found for the mathematical model. It should be noted that the maximum deviation in the mathematical prediction results (21.11%) occurred in the 5th testing experiment, which showed a high RD deviation . Furthermore, mathematical models also displayed some deviations exceeding 10%, contrary to ANFIS deviation values. The results shown in peak, E, and SEA. Desirability analysis was used to select the best settings of the CF incorporation, relative density, cell type, and cell size RD that led to maximizing the σStress-strain curves depicted in peak, and SEA. For instance, the maximum compressive modulus (0.549 GPa), σpeak (15.768 MPa), and SEA (15.591 J/g) were observed with Diamond, 15% CF, 44% RD, and 12 mm cell size (run #8). On the other hand, the worst mechanical responses were obtained by Primitive-based cell-type TPMS structures. The minimum compressive modulus (0.144 GPa) was obtained with Primitive, 0% CF, 30% RD, and 8 mm cell size (run #17), while the lowest σpeak (4.583 MPa) was observed with Primitive, 15% CF, 30% RD, and 8 mm cell size (run #21). Similarly, the minimum SEA (3.759 J/g) was observed with Primitive, 15% CF, 30% RD, and 12 mm cell size (run #22). The walls’ orientation and better material distribution within the geometry of the Diamond structures improve wall contact and reduce the empty spaces, making it less susceptible to fracture initiation [From the results presented in odulus 0.49 GPa, σitiation . For illpeak . The statistical analysis confirms that cell type and RD influence the compressive modulus, σpeak, and SEA of the TPMS lattice structures. This finding is in line with previous studies that investigated the effect of TPMS cell topologies and RDs on E, σpeak, and SEA characteristics, such that these properties were enhanced as RD increased [The influence of all considered parameters, including cell topology and size, CF incorporation, and RD, as well as their different combinations on the compressive response of the TPMS structures, was statistically significant. Results showed that RD had the greatest impact on mechanical response among the four studied variables, followed by cell type and CF incorporation. Cell size had the least impact. The authors of  stated tncreased . Diamondncreased . In thisCF incorporation was found to be a statistically significant influence on the mechanical properties and SEA. These findings are consistent with : CF-reinpeak was significant. Even though the combination of the three factors had no statistical influence on the SEA contrary to [Cell size had the smallest effect, and the statistical analysis confirmed its influence only on the compressive modulus. However, its interactions with cell type and RD significantly influenced the compressive modulus and strength, as seen in trary to   reported in ANOVA analysis  indicates a high error in mathematical modeling predictions for experiments having a high RD deviation. For instance, the higher mathematical modeling prediction deviations for σpeak  were observed for the testing experiments , with relatively high RD deviations; see peak, and SEA. A high correlation between the RD deviation and mathematical prediction deviation in the case of σpeak (R2 = 0.802) can be observed, while a low correlation is in the case of SEA. peak, which is highly influenced by the RD variable (71.68%). A correlation was observed between the contribution percentage of the RD influence on the TPMS structure performance , and SEA (3.759 J/g to 15.591 J/g) due to the impact of the considered variables.The findings demonstrated a change in the E (0.144 GPa to 0.549 GPa), σpeak, with a contribution of 58.95% and 71.68%, respectively. The cell type had the highest impact on the SEA, contributing to 70.51% of the total influence. In general, RD had the highest influence on mechanical responses among the four studied variables, followed by cell type, CF incorporation, and finally, cell size having the least impact.RD had a significant influence on both E and σThe findings revealed the importance of statistically evaluating the influence of the design, RD, and material composition  parameters and their combination to attain the desired TPMS lattice structure performance.peak, and SEA well. However, when it comes to the testing data set , ANFIS models clearly outperformed mathematical models in terms of RMSE and absolute percent deviation in predicting all mechanical characteristics. For instance, the maximum absolute percent deviation was 7.61% for ANFIS prediction, while it was 21.11% for mathematical prediction.For the training data set , both mathematical and ANFIS models predicted E, σpeak, there was a significant correlation (94.3%) between the accuracy of mathematical predictions and RD deviation. Therefore, for a high variation in RD, ANFIS models are preferable.The accuracy of mathematical predictions is highly influenced by the degree of RD deviation; a higher deviation in RD results in lower accuracy of predictions. Furthermore, a correlation between the mathematical models’ prediction accuracy and the RD deviation was found when the RD influence contribution on a TPMS performance was high. For instance, when RD accounted for 71.68% of the variation in σWhenever the actual RD is close to the designed RD, both models provide accurate prediction models. This also indicates the ability of both models to predict different mechanical properties of TPMS structures, taking into account different variables, including material composition, geometry, and the RD.peak, and SEA.This study provides a better understanding of PLA and CFRPLA TPMS structures, as well as an ability to better predict their mechanical behavior. For instance, based on the multi-objective optimization using the desirability approach, the Diamond topology, 12 mm cell size, 15% CF, and 44% RD combination are the best settings that achieved an overall desirability of 97.8% for E, σIn this study, the influence of different TPMS-based cell topologies with varying unit cell sizes, relative densities, and CF incorporation on the mechanical and specific energy absorption was statistically investigated. The FDM 3D-printed lattices were tested using a uniaxial compression testing, and their E, σ


To date, epidemiological studies have not evaluated heroin-related deaths in the Middle East and North African regions, especially Saudi Arabia. All heroin-related postmortem cases reported at the Jeddah Poison Control Center (JPCC) over a 10-year period (21 January 2008 to 31 July 2018) were reviewed. In addition, liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was utilized to determine the 6-monoacetylmorphine (6-MAM), 6-acetylcodeine (6-AC), morphine (MOR), and codeine contents in unhydrolyzed postmortem specimens. Ninety-seven heroin-related deaths were assessed in this study, and they represented 2% of the total postmortem cases at the JPCC . In the blood, urine, vitreous humor, and bile samples, the median morphine concentrations were 280 ng/mL, 1400 ng/mL, 90 ng/mL, and 2200 ng/mL, respectively; 6-MAM was detected in 60%, 100%, 99%, and 59% of the samples, respectively; and 6-AC was detected in 24%, 68%, 50%, and 30% of the samples, respectively. The highest number of deaths  was observed in the 21–30 age group. In addition, 61% of cases were classified as “rapid deaths,” while 24% were classified as “delayed deaths.” The majority (76%) of deaths were accidental; 7% were from suicide; 5% were from homicide; and 11% were undetermined. This is the first epidemiological study to investigate heroin-related fatalities in Saudi Arabia and the Middle East and North African region. The rate of heroin-related deaths in Jeddah remained stable but increased slightly at the end of the study period. Most patients were heroin-dependent abusers and from the middle-aged group. The availability of urine, vitreous humor, and bile specimens provided valuable information regarding the opioids that were administered and the survival time following heroin injection. The abuse of heroin  remains a major cause of death worldwide. For example, an estimated 62 million people used opioids for non-medical purposes in 2019 , with half using heroin or opium ,4, whileIn the Middle East and North African (MENA) region, principally in the Arabian Gulf region, heroin is reported in the media as a symbol of drug addiction. However, heroin-related fatalities have rarely been reported in scientific literature from this region. Thus, little is known about heroin-related deaths, and most studies in the Arabian Gulf region are outdated ,9. Al-MaIn Saudi Arabia, alcohol and drug use are prohibited by law and denounced based on social and religious perspectives ,12. ThusHeroin is unstable in biological specimens and rarely identified in postmortem cases . AlternaThe patterns of heroin-related deaths are poorly understood in the MENA region. Thus, up-to-date epidemiological studies to reveal heroin-related fatalities and explore the problem of heroin abuse are a crucial task. As Jeddah is one of the major cities in Saudi Arabia (4 million population), this paper reports the first epidemiological study evaluating heroin-related deaths in Jeddah City, Saudi Arabia, between 2008 and 2018 and investigates whether metabolites of heroin found in various bodily fluid samples can be used to identify the type of opioid ingested and the time of death after administration. Such toxicological information is crucial for providing knowledge on the trends in illegal drug use and planning initiatives to reduce accidents and deaths among drug users.® solid phase extraction (SPE) cartridges (CSDAU203) were obtained from United Chemical Technologies .Morphine, morphine-d3, 6-MAM, 6-monoacetylmorphine-d3 (6-MAM-d3), codeine, codeine-d3, and 6-AC were purchased from Lipomed . Methanol (HPLC grade), acetonitrile (HPLC grade), ammonium carbonate, formic acid, and ammonium hydroxide were obtained from BDH . Ammonium formate was obtained from Sigma-Aldrich . Clean Screen2O), and 2 mL of 0.1 M phosphate buffer adjusted to pH 6. The sample mixture was then loaded onto the SPE column using gravity. Next, the cartridges were washed by adding 1 mL of D.H2O, followed by 1 mL of acetic acid (0.1 M), and then dried under vacuum for 5 min. The third washing step was completed by adding 2 mL of hexane. Two elution steps were performed: the first elution (A) was performed by adding 2 mL hexane/ethyl acetate . Next, the elution tubes were removed, and the SPE cartridges were washed using 3 mL of methanol and then dried under full vacuum for 2 min. The second elution (B) was performed by adding 3 mL of dichloromethane/isopropanol/ammonium hydroxide  to each cartridge. Both fractions were collected in a glass evaporation tube and evaporated to dryness using nitrogen. Finally, 200 μL of the initial mobile phase was added to the final extracts and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) using an injection volume of 1.0 μL.One milliliter of each specimen was placed in a glass test tube, which was then spiked with 50 µL of the internal standard . The mixture was then mixed and vortexed for at least 10 s. Next, 2 mL of 0.1 M phosphate buffer (pH 6) was added to the samples, mixed, and centrifuged for 10 min at 3500 rpm. Before loading the samples into the SPE cartridges, the samples were prepared for SPE by adding 2 mL of methanol, 2 mL of deionized water  equipped with electrospray ionization (ESI+) and selective reaction monitoring modes. Analytes were separated using a Synergy Polar RP column  equipped with a guard column with identical packing material . The column oven and auto-sampler tray temperatures were kept at 30 °C and 4 °C, respectively. The gradient mobile phase consisted of ammonium formate buffer  as mobile phase A and an organic modifier of 100% acetonitrile as mobile phase B, with a flow rate of 0.3 mL/min for the whole run. The gradient program was initiated by applying 3% B for 3 min, which was increased to 15% over the next 5 min, 26% over the next 7 min, 80% over the next 13 min, and 95% over the next two min. After 27 min, the initial mobile phase was applied for 3 min. Data were acquired and managed using the Xcalibur system .The second LC-MS/MS analysis was performed according to a previously reported method . Brifly,This study was approved by the IRB committee of Jeddah Health Affairs, Ministry of Health, Jeddah, Saudi Arabia .Blood samples were collected from the subclavian site in tubes containing 1% sodium fluoride (BNaF). Vitreous humor fluid was collected in gray tubes containing sodium fluoride, and urine and bile samples were stored in a plain container without any preservative. All samples were stored frozen (at −20 °C) until analysis. Autopsy samples were thawed to obtain them and refrozen until use. Blood samples were obtained for 84 postmortem cases (87%), urine samples were obtained for 74 cases (76%), vitreous humor samples were obtained from two eyes in 70 cases (72%), and bile specimens were obtained for 27 cases (28%).All postmortem body fluids and tissues collected for forensic toxicology investigations are analyzed for commonly abused drugs and reported, and they are also assessed based on requests by forensic pathologists. All heroin-related postmortem cases reported at the Jeddah Poison Control and Medical Chemistry Center (JPCC) over the last 10-years (21 January 2008 to 31 July 2018) were reviewed. The 6-MAM, 6-AC, morphine, and codeine contents were reviewed, and case details were collected from the online Forensic Toxicology Jeddah Reports Database. The search was conducted between February 2015 and July 2018. Data before February 2015 was manually collected from JPCC archive files. Cases positive for heroin use were reviewed and included in this investigation according to the inclusion/exclusion criteria mentioned below. Ninety-seven cases reported during the study period were included.The same criteria applied in our previous study were used in the current study . The mosAll postmortem cases, irrespective of the manner of death, were screened using a common immunoassay reagent. The second step was to search for other concomitant drugs using general unknown screening approaches (GUS),  by gas chromatography (GC) coupled to a flame ionization detector (FID), gas chromatography coupled to mass spectrometry (GC-MS), and LC-MS/MS.The analysis methods were all fully validated using international guidelines for forensic investigation , and a sp-value <0.05 was considered statistically significant.Useful statistical data are reported in terms of the frequency, percentage, mean, median, and range when applicable. The data were calculated using Statistical Packages for Software Sciences (SPSS) version 28.0.1.1  and Microsoft Excel version 16.66.1 . Descriptive statistics were completed, and continuous data were presented as median, minimum, and maximum. Definite data were displayed as frequency and percentage. A Mann-Whitney U test was employed to estimate variations between groups. Spearman’s correlation test (R) was utilized to compare variables. A The method in the current investigation was validated according to ANSI/ASB standards  and otheHeroin biomarkers are not stable, which leads to a decrease in the concentration of these biomarkers in tested specimens that are not stored properly; therefore, a sensitive method is required to measure these biomarkers. The stability of these heroin biomarkers is a well-known issue that must be considered during sample processing, namely, during sample storage, preparation, and analysis in an autosampler. Therefore, all specimens were immediately frozen, thawed before extraction, and then immediately refrozen after sampling. The stability of these heroin biomarkers was investigated in an autosampler using three controls that were previously used in precision studies, and they were reanalyzed after 24 h, 48 h, and one week.The most important feature of such analysis methods is to investigate the limit of detection (LOD) in the proposed matrix of interest and the workable lower limit of quantification (LOQ) that can be utilized to quantify these unstable analytes at very low concentrations (≤1 ng/mL). Elevated morphine concentrations in urine and bile were used as the upper limit of quantification (ULOQ) and assessed for all heroin-related analytes. The sensitivity analysis was performed in accordance with the ANSI/ASB method validation guidelines .In each batch of samples, negative blank samples from different matrices were included without any standards or internal standards. Negative blank samples with internal standards were only used to investigate method selectivity, and negative blank samples were run following the calibrator with the highest concentration to investigate any carryover. Matrix effects were investigated using post-extraction addition as reported by Matuszewski et al. . Six difThe validated analysis method was acceptable , and theLinearity was accepted, with coefficients of determination greater than 0.99 for 6-MAM, 6-AC, morphine, and codeine in multiple body fluid samples. The LOD values were estimated from ten different calibration curves and ranged between 0.2 and 0.4 ng/g, while the LOQ was evaluated by spiking with 1 ng/mL of each analyte of interest, and the results were calculated using freshly prepared calibration curves for all analytes of interest in different matrices of interest. The precision, accuracy, dilution, and autosampler stability results were all within 15% of the nominal value, thus confirming that the method is suitable for the quantification of target analytes in the matrices of interest. In addition, carryover contamination from the previous positive control test was not detected.n = 85; range, 23–4400 ng/mL); the highest median BNaF morphine concentration occurred in 2012 (470 ng/mL), while the lowest median concentration occurred in 2008 (141 ng/mL).The number of heroin-related deaths in the city of Jeddah within the study period represents 2% of the total number of postmortem cases received by the JPCC between 2008 and 2018 . AlthougThe mean age of the patients was 38 ± 12 years . The 21–30 age group had the highest number of deaths, with almost 33% of the total cases (n = 32 cases), followed by the 31–40 age group , while the 10–20 age group had the lowest number of heroin-related death cases , which led to the identification of both heroin biomarkers when the blood was fresh and putrefaction was not observed. The median concentrations of 6-MAM and 6-AC in cases with a PMI within 24 h were 16 and 2 ng/mL, 22 and 3 ng/mL, 324 and 35 ng/mL, and 12 and 4 ng/mL in the BNaF, vitreous humor, urine, and bile samples, respectively . 6-AC waIn the current study, both biomarkers were detected in the vitreous humor in two cases with a PMI longer than 10 days, whereas 6-MAM was negative in the BNaF samples, which indicates that the role of PMIs on heroin biomarkers can be minimized if samples are stored correctly following autopsy and if the vitreous humor is the sample of choice for heroin-related fatality investigations. In the current study, heroin alone contributed to death in 68% of the studied cases, whereas intoxication with other co-ingested substances contributed to 32% . A higheThese findings indicate that the morphine concentration in heroin-related fatalities showing the presence of other CNS drugs is often lower than that in heroin-alone cases. The significant role of the PMI in cases of heroin metabolites is well known, and the tolerance and health status of the deceased contribute to this finding. Similar trends were observed among the various specimens, with slightly higher median concentrations in the urine and bile. Moreover, a polydrug intoxication case showed a slightly higher morphine concentration in the vitreous humor than that with heroin alone.The most frequently detected drugs, regardless of the cause of death, were methamphetamine, cannabis, amphetamine, alprazolam, cocaine, and ethanol. Thirty-eight cases only showed heroin metabolites. A higher median morphine concentration was measured in only heroin cases compared to those in which heroin was the sole cause of death despite the presence of another drug, such as amphetamine or cannabis (370 ng/mL vs. 310 ng/mL). Similarly, the median morphine concentration (312 ng/mL) decreased when one extra drug was used, and it was further decreased (191 and 198 ng/mL) when two and four extra drugs were co-ingested, respectively.Notably, methamphetamine was co-ingested with heroin in twenty-three cases . The median morphine concentration for most co-ingested drugs was often higher than 200 ng/mL. A reduction in the median morphine concentration  occurred when heroin was used in combination with cocaine.In this study, 61% and 24% of the cases were rapid and delayed deaths, respectively, and 15% had an undetermined time of death. This was either because of a lack of information caused by the deceased dying without a witness or because the bodies were moved after death to a deserted area where the bodies began to decompose and thus did not show signs of heroin use, such as injection marks . In thesThis suggests that either the undetermined cases died immediately after heroin was administered and the environmental surroundings allowed for the hydrolysis of 6-MAM to morphine or that a delayed death occurred, which allowed morphine glucuronide to deconjugate to free morphine due to the long PMI before sampling. In contrast, lower median morphine concentrations were observed in the delayed death group (median BNaF = 210 ng/mL) than the rapid death group.In all groups, the median morphine concentration was much lower in the vitreous humor than the blood. This indicates that some of the heroin biomarkers were converted to their metabolites in the blood but were stable in the vitreous humor after death. Nevertheless, the vitreous humor results should be interpreted with caution because a certain amount of morphine is associated with accumulation from the use of old doses by chronic heroin users. Interestingly, 6-MAM levels were higher for the rapid death cases (36 ng/mL) than the delayed death cases (14 ng/mL) and undetermined death cases (7 ng/mL). This can be explained by many factors, including the longer PMI in delayed deaths, putrefaction effects, and body storage effects, especially when the time of death is unknown. These conditions led to a decrease in the 6-MAM concentration in the vitreous humor. This highlights the importance of testing the vitreous humor in cases of longer PMIs and negative 6-MAM in the blood.Morphine levels in bile were primarily detected in chronic heroin users, which limits the use of these values in distinguishing between rapid and delayed deaths. 6-MAM in bile was detected in 10 of 17 rapid deaths, 3 of 6 delayed deaths, and not detected of 4 undermined cases. Urine is most likely to be positive for heroin biomarkers and their metabolites in these types of deaths. In this study, the free morphine concentration was high for rapid deaths (2100 ng/mL) compared to that for delayed deaths (1300 ng/mL) and undetermined cases (550 ng/mL). An almost 4-fold higher median concentration of 6-AC in urine was observed for rapid death (40 ng/mL) compared with delayed death (10 ng/mL). Moreover, the median codeine concentration was higher in the unknown group (30 ng/mL) than in the rapid (20 ng/mL) and delayed groups (10 ng/mL). Codeine forms quickly after heroin ingestion as a product of 6-AC degradation. Most codeine is metabolized to codeine-6-glucuronide, while some is metabolized to morphine.In the current study, 76% of the total cases were ascribed to accidental death, and they had a median age, median BNaF morphine concentration, and PMI of 38 years, 280 ng/mL, and 24 h, respectively. Nevertheless, most heroin abusers in Saudi Arabia used drugs in private or remote areas, such as deserted areas and open land outside the city. In the case of overdoses, deceased bodies left behind without witnesses complicated the identification of the mode of death, especially if the bodies were heavily putrefied.Information about intentional overdoses was available for seven heroin-related fatalities. Four of these individuals died at home, and two died outdoors while accompanied by a friend. These cases had a median BNaF morphine concentration of 480 ng/mL, a PMI of 24 h, and an age of 48. The highest levels of vitreous humor and morphine were detected in suicide cases (150 ng/m).In five cases, the mode of death was identified as homicide, and they presented a median BNaF morphine concentration of 180 ng/mL, a PMI of 24 h, and an age of 22. Notably, most cases were discovered outdoors.The data in Most heroin deaths occurred in outdoor environments (63%), with a median PMI of 48 h, whereas 37% of the patients died in a private home , with a It has been mentioned that the weather in Saudi Arabia is extremely hot most of the year, and the weather in Jeddah is known to be highly humid. Accordingly, it is well-known that putrefaction and postmortem changes would begin a few hours following death if bodies were not stored appropriately ,33. In cIn 63 out of 97 cases (65%), police and forensic pathologist reports indicated that no witnesses observed the final moments before death . Nine paIn this study, higher BNaF morphine concentrations were measured in patients whose deaths were pronounced at hospitals , followed by those found in the street , under bridges , in cars , in rental flats or hotel rooms , and in deserted areas . PMI, environmental conditions (location of death and weather), and patient tolerance have major effects on the detection of heroin biomarkers and metabolites. The patients who died at the hospital had a lower PMI, and their deaths were caused by an overdose of heroin alone. In contrast, among individuals who died under bridges, most cases showed putrefaction, and 6 out of 10 cases were polydrug intoxication cases. Notably, a high number of deaths occurred in cars, with 13 out of 17 cases of polydrug intoxication having a relatively low PMI (median 24 h) and 6 cases exhibiting signs of putrefaction, with two heavily putrefied. Vitreous humor and urine were available in 13 and 12 of these cases, respectively, and 6-MAM and 6-AC were detected in 6 and 9 cases, respectively. Only one case was positive for both heroin biomarkers in bile. A higher median of BNaF morphine was observed compared to vitreous humor (60 ng/mL), which suggested a shorter survival time. This was supported by the lower urine morphine concentration (860 ng/mL) and highest bile morphine concentration (9800 ng/mL), suggesting a chronic heroin user.Twenty-one cases showed signs of putrefaction , 60% shoThe seasonal distribution of heroin-related fatalities is presented in The median morphine concentrations in the BNaF, urine, vitreous humor, and bile samples were 280, 1400, 90, and 2200 ng/mL, respectively. Among the available BNaF, urine, vitreous humor, and bile specimens, 6-MAM was detected in 60%, 100%, 99%, and 59%, while 6-AC was detected in 24%, 68%, 50%, and 30%, respectively. The presence of sodium fluoride in the blood test tube as a preservative prevented 6-MAM from converting to morphine in most cases. Additionally, the availability of urine, bile, and vitreous humor samples provides valuable information on the opioids that have been administered. The median free morphine concentration ratios among the vitreous humor/BNaF, urine/BNaF, and bile/BNaF were 0.37-fold (n = 63), 5.6-fold (n = 64), and 7.3-fold (n = 23), respectively. In addition, free morphine/free codeine were always higher than 1, with median ratios of 13-fold (n = 84), 13-fold (n = 70), 6-fold (n = 68), and 85-fold (n = 25) for BNaF, urine, vitreous humor, and bile, respectively.p-values higher than 0.5). The correlation between the 6-AC concentration obtained from multiple biological fluids was always poor and not statistically significant, and it was not calculated for bile due to the lack of positive samples. A negative correlation was observed between 6-AC and morphine in bile and analytes of interest in the BNaF, urine, and vitreous humor samples, as shown in The Spearman correlation coefficient (R) was used to establish the relationship between heroin biomarkers, morphine, and codeine concentrations in various body fluids . 6-MAM iGood correlations were obtained between the analytes of interest detected in BNaF vs. vitreous humor, BNAF vs. urine, and vitreous humor vs. urine. The level of 6-AC seemed to be higher in the vitreous humor than the blood, which can directly reflect the anatomical location of the vitreous humor; thus, 6-AC in the vitreous humor is more stable and has a longer half-life before conversion to codeine than that in blood samples.The bile samples showed the lowest positive results for 6-AC (eight cases) compared to 6-MAM. The 6-MAM was detected in 16 of the 27 bile samples tested in this study. Although the correlation between heroin-related compounds in BNaF and bile was weak, the correlation between these analytes in bile and their corresponding specimens in vitreous humor and urine was strong, except for the 6-AC concentration.In the literature, few studies have detailed the relationship between addiction and morphine concentration. Steentoft et al. reported 245 heroin-related deaths, and all of these cases were from known heroin abusers with the exception of 13 cases . ElfawalHeroin users often die young because of polydrug intoxication , and mosp > 0.05) [Jones and Ahlner studied 766 postmortem heroin-related deaths and 124 traffic antemortem cases related to heroin use and found no correlation between the concentration of free morphine and the age of heroin users  . It is bThe level of codeine was low in all age groups , which could be explained by the fact that 6-AC is metabolized to codeine and codeine-6-glucuronide (C6G) faster than to other compounds. A recently published study demonstrated that C6G was not detected in blood samples obtained from authentic postmortem cases following heroin intake, while codeine was . In anotSkopp concluded that the PMI competes with the postmortem redistribution phenomenon (PMR) , and theFugelstad et al. studied the effects of postmortem changes on heroin-related fatalities and concluded that the concentration detected at autopsy was the same as that at the time of death . In contPMR has been debated scientifically, especially for postmortem blood morphine concentrations following heroin-related deaths. Maskell et al. suggested the use of vitreous fluid to verify heroin use because minimal morphine change was detected in their study . An incrThe use of blood-free morphine/total morphine ratios in the blood to identify the cause and mode of death has been reported ,42. HoweJones and Ahlner found no differences in the median blood morphine concentration between intoxication with polydrug and heroin-only . MeissneThis study is consistent with a previous investigation that found lower morphine concentrations when cocaine was co-administered . The lowIn this study, few case were positive for heroin and ethanol, where ethanol was attributed to antemortem ethanol ingestion. Moreover, the few cases may have been related to the low sample size of heroin users and the prohibition on ethanol intake by law in Saudi Arabia due to religious reasons ,60. In cNotably, In 2018, alprazolam was detected in 4 out of 15 cases; most of these cases involved polydrug intoxication, including methamphetamine, cocaine, and tramadol. In Saudi Arabia, alprazolam is a controlled drug, although the alprazolam supply has increased in illegal markets, which explains this increase. A similar trend has been observed with methamphetamine-related postmortem deaths in Jeddah, Saudi Arabia, in the same period . Abuse oConsistent with previous reports, deaths following heroin intake can be divided into three types: rapid and delayed deaths, and undetermined survival times ,3,51. RaGoldberger et al. reported similar findings to the current study and showed that a higher median free morphine concentration occurred for rapid deaths 1420 ng/mL) compared to delayed deaths (250 ng/mL), while the median morphine for undetermined survival time after ingestion (610 ng/mL) was higher than that for delayed deaths . Recentl0 ng/mL cThe concentration of morphine in the vitreous humor is often lower than that in the blood. However, this concentration was found to be dependent on the survival time following heroin administration.The use of the bile free morphine concentration to distinguish between rapid and delayed deaths has received little attention, and in most cases, the total morphine content has been reported. The total morphine concentration is significantly higher with delayed deaths, which indicates that the deceased is heroin-dependent and has received high doses of heroin . The samIn general, heroin-related deaths are most likely to occur due to accidental overdoses, although suicidal or homicidal modes of death have also been reported in the literature ,69. AbirDepression is an indicator associated with heroin abuse, and it has led to an increase in violence-related deaths among heroin users ,72. DarkOpioids, such as heroin, are sedative drugs that are rarely associated with violence ,73,74. IThe median morphine concentration reported in this study was considered higher in cases of suicide-related deaths compared to those of accidental intoxication, which is consistent with the study by Darke et al. . In mostHeroin overdose as a means of homicide has been reported, and the percentages of heroin-related homicide have varied between studies. For example, in a study by Denninng et al., almost 20% of cases were reported to be homicides by drug overdose . In otheOur findings were consistent with the study by Thiblin et al.  . A higheIn the current study, no differences were observed in the median vitreous humor concentration among the three routes of administration. The most useful information obtained by measuring vitreous humor is the source of opiates administered when 6-MAM is detected in this matrix . In contIn a study by Fugelstad et al. , patientIt has been widely reported that heroin addiction is one of the main causes of HIV, and therefore, we believe that it is important to discuss the number of individuals who died from HIV. As the discussion here pertains to an epidemiological investigation in certain parts of the world for the first time, this piece of information is important. However, one of the limitations of this study is that the infectious disease history was not detailed in all cases. HIV and hepatitis were reported in fewer than 1% and 3% of total cases, respectively, which may indicate that sharing needles is not a common practice or that HIV deaths related to heroin addiction are under-reported in Saudi Arabia, considering that deaths due to infectious diseases are not usually subjected to postmortem analysis.The location where the bodies were found following a drug overdose is an important factor when interpreting the cause and manner of death. The impacts of ecological conditions, such as temperature and humidity, and location, such as indoor and outdoor places, on the analytes of interest in the postmortem samples were investigated. Few studies have discussed the location of heroin-related deaths. Fugelstad et al.  found thIn previous studies, although putrefaction was reported, limited information regarding heroin-related metabolites was available. In the study by Crandall et al., putrefaction was observed in 12% of heroin-related deaths . Crump eThe impact of seasonal variation on heroin biomarkers and their metabolite analysis is believed to be a crucial factor underlying heroin-related deaths, especially in countries such as Saudi Arabia, where hot weather is more dominant. Kringsholm et al.  reportedIn forensic postmortem procedures, the ratio of morphine to codeine is of paramount importance in differentiating the source of opioid ingestion. A morphine-to-codeine ratio of less than one is considered an indicator of the intake of medicine containing codeine, while a morphine-to-codeine ratio of more than one is related to the use of heroin ,51,92. Tr2 = 0.01, p = 0.4), which agrees with the current investigation. In that study, the median ratio of free morphine between the bile and blood was twenty-nine, which was lower than that in the current study. Duflou et al. reported the total morphine in blood and bile, and a strong correlation for the total morphine between blood and bile was obtained when using their data, with Rs = 0.712 and a p-value of 3 × 10−5, and the median ratio of free morphine between bile and blood was 37 [Many factors that affect the connections between heroin biomarkers and their metabolites must be taken into consideration, such as the history of abuse, concomitant use of other drugs, tolerance, route of administration, and PMI ,33,44. Md was 37 . The mord was 37 . Negativd was 37 . A low md was 37 .2 = 0.697) in all cases, whereas a strong correlation was obtained in 17 cases when the deaths were described as sudden (r2 = 0.885). The same observation was reported by Rees et al. [p = 8.0 × 10−7) than delayed death cases .The correlation of morphine in the blood with the vitreous humor has been widely studied. Scott and Oliver  found ths et al. , who fouTo the best of our knowledge, this is the first epidemiological study evaluating heroin-related deaths in Jeddah, Saudi Arabia. Although the weather in Saudi Arabia is extremely hot most of the year, 6-MAM hydrolysis to morphine is favored. The detection of heroin biomarkers (6-MAM and 6-AC) is facilitated by short PMI periods in the majority of cases; moreover, the addition of sodium fluoride as a preservative prevents 6-MAM from being degraded to morphine in most cases. In addition, the availability of urine, vitreous humor, and bile samples provides valuable information on which opioids have been administered based on the detection of heroin biomarkers. The latter is important if no blood samples are available, if blood samples are negative for heroin biomarkers, and in cases of partially and heavily decomposed bodies. The incidence of heroin deaths in Jeddah remained stable during the study period. Most individual deaths likely occurred within groups, but the body was left behind or moved owing to fear among users to call an ambulance. Although only 24% of the deceased had medical records, the findings indicated an improvement in health reporting regarding drug abuse in Saudi Arabia. It is highly recommended that heroin users be encouraged to contact ambulances and trained in resuscitation. This study concluded that most patients were unemployed (up to 53%), had started taking heroin in their teens, and had been admitted several times to the addiction center. Therefore, the loss of tolerance following detoxification might have been the primary cause of many heroin-related deaths.


Olea europaea L.) fruit reduces the sensory and nutritional qualities of olive oil, thereby increasing production costs. Polyphenol oxidases (PPOs) are the key enzymes that catalyze phenolic substance oxidation and mediate enzymatic browning in olive fruit, but the exact regulatory mechanism remains unclear. The main challenge is the lack of comprehensive information on OePPOs at the genome-wide level. In this study, 18 OePPO genes were identified. Subsequently, we performed a bioinformatic analysis on them. We also analyzed the expression patterns and determined the relationship among browning degree, PPO activity, and expression of OePPOs in the fruits of three olive varieties. Based on our analysis, we identified the four most conserved motifs. OePPOs were classified into two groups, with OePPOs from Group 1 showing only diphenolase activity and OePPOs from Group 2 exhibiting both mono-/diphenolase activities. Seven pairs of gene duplication events were identified, and purifying selection was found to have played a critical role in the evolution of the OePPO gene family. A positive correlation was observed between the browning degree of olive fruit and PPO activity across different olive varieties. Moreover, two important genes were found: OePPO-5 the main effector gene responsible for fruit browning, and OePPO-8, a key gene associated with specialized metabolite synthesis in the olive fruit. In short, our discoveries provide a basis for additional functional studies on OePPO genes and can help elucidate the mechanism of enzymatic browning in olive fruit in the future.Browning of olive ( Olea europaea L.) is an oil crop belonging to the Oleaceae family and Olea genus. The extraction of olive oil from olive fruit results in a product that is rich in nutrients and bioactive compounds [The olive plant  are a type of metalloenzyme that contains a type III copper center. They are widely found in archaea, bacteria, fungi, plants, and animals ,4. It isIn plants, PPOs are typically expressed in an inactive form as proenzymes, with a molecular weight of 54–62 kDa. This proenzyme form of PPOs includes an N-terminal transit peptide of approximately 8–12 kDa and a C-terminal domain of around 15–20 kDa . Apart fBrowning is a common phenomenon in plants, and PPOs are well-known for their involvement in the browning reaction. In normally growing plants, PPOs are typically localized in chloroplasts ,19. PPOsIn this study, OePPOs were identified from the entire olive genome, and their gene structures, phylogenetic relationships, chromosomal locations, and collinearity were analyzed. Furthermore, the expression patterns of these PPOs in different tissues of the olive plant were investigated. Herein, we aimed to predict the types (TYRs or COs) of the 18 identified olive PPOs (OePPOs), determine the relationship between browning degree and PPO activity in different olive fruit varieties, and identify the main effector genes involved in browning within the OePPO family.PPO genes was obtained using Oxford Nanopore third-generation sequencing and Hi-C technology by our laboratory team and has been submitted to the China National Center for Bioinformation  [http://hmmer.org/, accessed on 1 February 2023) tool was employed to search for PPO protein sequences in olives. The sequences obtained from the two methods were merged, following which all candidate sequences were submitted to an NCBI Batch CD-search  to identify their conserved domains. After manual screening, 18 sequences were considered to be PPO family members.The high-quality olive genome CRA003087) used to identify the ry 2023) . A local87 used try 2023)  as the qhttps://services.healthtech.dtu.dk/services/TMHMM-2.0/, accessed on 1 February 2023) and ExPASy  were used to calculate the transmembrane domains (TMHs), theoretical isoelectric points (Pis), and molecular weights (MWs) of the olive PPO proteins.The online tools TMHMM . Through blastp and annotation information, we identified their polyphenol oxidase families. Subsequently, comprehensive comparisons of these PPOs and OePPOs were performed.At the same time, the protein data for The amino acid sequences of the OePPOs were analyzed by the MEME program to identify conservative motifs . The gffThe upstream 2000-bp regions of OePPOs were submitted to PlantCARE to identify cis-acting elements .The location information of the genes on the chromosome was extracted from the gff file using Tbtools . Tandem https://www.ebi.ac.uk/ena/browser/home, accessed on 1 February 2023), with the accession numbers PRJNA590386  and PRJNA596876 . Our laboratory team analyzed the data [To explore the expression patterns of OePPOs in different tissues, the original RNA-seq data in fastq format were obtained from the ebi-ena database , Arbosana (F2), and Koroneiki (F3). The fruit samples were added to pre-cooled phosphate buffer  in a ratio of 1:10 (w:w). The samples were then ground using a micro ball mill GT3000  at 1500 rpm for 1 min. Then, the samples were centrifuged at 4 °C and 12,000 rpm for 15 min. The supernatant was collected and used as the PPO enzyme source. 416 represents the browning degree.Browning degree assay: The supernatant was incubated in a water bath at 37 °C for 15 min. After incubation, the absorbance (A) was measured at 416 nm wavelength. The value of AEnzyme activity assay: The enzyme activity assay system consisted of 1 mL, including 800 μL phosphate buffer, 100 μL 0.1 mol/L catechol, and 100 μL enzyme source. The absorbance was recorded at 400 nm every 30 s for a total of 3 min. Under the measurement conditions, a change of 0.01 in A400 per minute per gram of sample represents one unit of polyphenol oxidase activity {U/(g·min)}.Excel 2021 was used to perform the statistical analysis for all the data, calculate the standard deviation, and create graphs. The significance analysis was conducted using SPSS 26.OePPOs were selected through local blastp and screened based on the presence of the tyrosinase domain structure , 4-methylcatechol , dihydroxyphe nylacetic acid acetic acid), dihy-droxyphenylalanine -2-amino-3-propanoic acid), and dopamine benzene-1,2-diol), but they do not have catalytic activity towards monophenols [To further investigate the potential functional divergence of OePPOs, a phylogenetic tree was constructed by performing a systematic evolutionary analysis of the TYR domains of the 18 OePPOs along with the TYR domains of other plant PPOs whose activities have been previously studied. The species include vinifera  . In contrast, in OePPO-15 and OePPO-17, the proportion of defense and stress responsiveness elements is the highest, accounting for 47.35% and 50%, respectively, which is higher than the second highest proportion of light responsiveness elements . This result suggests that OePPO-15 and OePPO-17 may play a major role in resisting environmental stress and adversity, similar to some previously reported plant PPOs [However, in the OePPO-1 and OePPO-2 are located on Contig01205 and Contig001268, respectively, while the rest of the OePPO genes are located on chromosomes  to the number of synonymous substitutions per synonymous site (Ks). As shown in OePPOs range from 0.15 to 0.58, with an average Ka/Ks value of 0.33. These results suggest that purifying selection may have played a critical role in the evolution of this gene family.Based on the above results, it can be observed that OePPOs, their RNA-seq expression patterns were quantified based on the FPKM values in six different tissues   A and thrd stems) B. FigureOePPOs also exhibit differential expression patterns in fruits, new leaves, and old leaves. Based on the clustering pattern in the figure, it can be observed that OePPO-5 and OePPO-8 are highly expressed in both fruits and new leaves, OePPO-9 is mainly expressed in fruits, and OePPO-3, 4, 7, 10, 11, 12, 14, 15, and OePPO-16 are mainly expressed in new leaves. Similarly, the genes predominantly expressed in each tissue are not exclusively classified into Group 1 or Group 2 but are evenly distributed between the two groups. In fruits, OePPO-5 and OePPO-9 belong to Group 1 and Class III, while OePPO-8 belongs to Group 2 and Class IV. In new leaves, OePPO-3, 4, 7, and OePPO-15 belong to Group 1 and Class III, while OePPO-10, 11, 12, 14, and OePPO-16 belong to Group 2 and Class IV. Genes from both Group 1 and Group 2 are uniformly expressed in each tissue.The main economic value of the olive plant comes from olive oil production, meaning its economic value is primarily in its fruits. However, olive fruits have a soft texture and are susceptible to squeezing and collisions during harvesting, transportation, and processing, leading to mechanical damage and browning.In this study, three dwarf and high-density olive varieties were used as materials to investigate the differences in browning when subjected to mechanical damage . Among them, Physcomitrella patens has the highest number of PPO genes, with 13 PPO genes identified. Arabidopsis thaliana does not contain any PPO genes [Arabidopsis thaliana indicates that the gene repertoire of certain species is mainly a result of independent bursts of gene duplication [PPO genes in olives, which is much higher in number compared to most plants, suggesting that the PPO gene family plays a crucial role in the growth, development, and metabolism of olives. Olives are rich in characteristic metabolites such as oleuropein and hydroxytyrosol, and PPOs are believed to be involved in the synthesis of these related metabolites [PPO genes in olives compared to many other species is likely associated with the synthesis of its characteristic metabolites.Through the local blast and tyrosinase domain structure, we identified 18 candidate d Taylor  researchPO genes , and it PO genes . The loslication . Similarabolites ,29. The OePPO-8, 10-14, 16, and 17, may be the key genes involved in mediating oleuropein synthesis. Moreover, considering the gene expression data in fruits  and OePPOs from Group 2 exhibiting both mono-/diphenolase activities (TYRs). The four most conserved motifs, motifs 5, 6, 9, and 12, are believed to be associated with differentiating COs and TYRs. Different olive fruit varieties were analyzed for browning degree and PPO activity. The results revealed a positive correlation between browning degree and PPO activity, indicating that PPO plays a crucial role in the enzymatic browning process of olive fruit. Furthermore, based on the expression levels, OePPO-5, which exhibits only diphenolase activity, was predicted to play a critical role in the browning process. Additionally, OePPO-8, which exhibits monophenolase activity, was predicted to be involved in the synthesis of specialized metabolites such as hydroxytyrosol and oleuropein. These findings provide a basis for further functional studies on the OePPO genes and to elucidate the mechanism of enzymatic browning in olive fruit, which could contribute to a reduction in the browning sensitivity of olive fruits in the future.In conclusion, a total of 18


Nannochloris sp., using cheese whey obtained as a side flow from cheese production as an organic carbon source. The microalgae samples were prepared by adding to the standard growth medium increasing amounts of cheese whey, calculated to ensure a lactose concentration between 0 and 10 g/L. The samples were incubated at a constant temperature of 28 °C and 175 rpm stirring speed for a total time of seven days. Two LED (Light Emitting Diode) illumination schemes were applied in order to assess the effect of this parameter on microalgae development and bioactive compound accumulation: continuous illumination (light stress) versus alternative cycles of 12 h light—12 h dark (day–night cycle). The growth medium was analyzed before and after microalgae cultivation in order to determine the reduction of carbon, nitrogen, and phosphorus. The results obtained for this process, after a seven-day cultivation period, were as follows: reduction of 99–100% of lactose from the growth medium, up to 96% reduction in chemical oxygen demand, up to 91% reduction in nitrogen content, and up to 70% reduction in phosphorus content.This paper focuses on a process for dairy wastewater treatment by mixotrophic cultivation of microalgae  Research results to date have shown that biomass produced by microalgae can be significant, both quantitatively and qualitatively ,2. TheseThe most promising biotechnologies refer to the use of microalgae for the production of biodiesel, biohydrogen (using anaerobic digestion), biogas, bioethanol, bio-methanol, bio-plastics, bio-fertilizers, medicinal products, and animal feed ,3,11. HoAnother use for microalgae systems is in wastewater treatment, where microalgae provide dissolved oxygen for bacterial degradation of organic waste, as well as causing a decrease in soluble nutrient concentrations. Microalgae biomass resulting from wastewater treatment could be transformed into co-products, for example, bio-fertilizers and raw materials for biofuels ,19.2, N, P, etc.), conventional methods mainly require long working times, large operating space due to multiple stages of treatment, and high energy consumption [2 in the environment during photosynthesis [The dairy industry generates large volumes of wastewater with a high content of nutrients, where microalgae epuration systems can be used. Although there are several methods for treating wastewater rich in nutrients  was studied at different volume ratios of dairy wastewaters to the blue-green growth medium, commonly referred to as BG-11. Following the experiments, chemical oxygen demand (COD) showed a range of variations from 62.5 to 87.5% for elimination efficiency. All microalgae strains used by Brar et al. [Brar et al.  investigr et al.  showed aChlorella vulgaris, Tetradesmus obliquus, and Nannochloropsis oceanica. The consortium was found to be more effective than the respective monocultures in recovering nutrients from the wastewater, achieving 88% removal of protein while completely removing nitrate and phosphate from the effluent within 14 days. In terms of biomass production, the best result was obtained by the single culture of Chlorella vulgaris, followed closely by the consortium.In a recent study, Kiani et al.  investigScenedesmus sp. for 11 days in photobioreactors (PBR), day–night cycle (12 h/12 h), both in the specific cultivation medium (BG11) as well as in 100% dairy industry wastewater (cheese and lactic derivatives production) showing a change in the biomass composition in the case of cultivation in wastewater from the dairy industry, which acts as a stress factor, increasing the concentration of lipids and decreasing the protein content. The cultivation of Scenedesmus sp. with 100% wastewater from the dairy industry also led to an 88% reduction in total nitrogen, 97% in total phosphorus, and 89% in COD.Mercado et al.  studied Nannochloris sp. microalgae strain in the removal of contaminants from dairy industry wastewaters, namely cheese whey effluents, used as an organic carbon source for mixotrophic cultivation. A first element of novelty for this study is the use of the strain Nannochloris sp. for dairy wastewater treatment, an often-overlooked microalgae strain, despite its high lipid productivity, which could offset some of the costs [Nannochloris sp. microalgae cultivation on dairy wastewaters and the effect it has on its metabolic activity.The goal of this study was to demonstrate the efficiency of the he costs . Researche costs ,29. Thishe costs . AnotherThe cheese whey used for the experimentations was obtained from a dairy farm where cow milk is used to prepare “Telemea”, a Romanian-specific cheese similar in appearance to feta cheese. Before being used for sample preparation, the cheese whey was submitted to a pretreatment stage. It was deproteinized, by precipitation, in order to avoid inhibition of the microalgae growth process. Deproteinization was performed by maintaining the cheese whey volume at boiling temperature for 15 min, with continuous stirring, followed by gradual cooling until room temperature, to facilitate precipitation, centrifugation, and filtration of suspended solids . The depTo determine initial lactose concentration, 0.2 mL of the sample solution was diluted up to 2 mL, followed by adding 1 mL of a 3,5-Dinitrosalicylic acid (DNSA) reagent. For determining the final lactose concentration, 2 mL of the sample solution was taken, followed by the addition of 1 mL of the DNSA reagent. The mixture was heated in a hot water bath at 95 °C for 5 min and then immediately cooled using cold water. The solution was analyzed using a UV-spectrophotometer -based DNSA assay for reducing sugars performed at 540 nm . Total nNannochloris sp. 424-1 is a proprietary patented microalgae strain [e strain , depositNannochloris sp., was a modified Zarrouk medium [Deproteinized cheese whey (DCW), priorly characterized, was used for culture medium preparation. The standard culture medium for the microalgae strain selected for these experiments, k medium , which hk medium ,35, withNannochloris sp. microalgae strain required for sample inoculation was prepared in advance in Erlenmeyer flasks, for 8 to 10 days of development time, at an ambient temperature of 21 ± 1 °C until the exponential growth phase of the microalgae strain was reached. Constant spectrophotometric monitoring of suspension samples at a wavelength of 678 nm allowed the identification of the point at which the microalgae cells pass from the inductive phase to the exponential growth phase. After reaching the exponential phase, the inoculum was used directly  for inoculating the control, and DCW-modified samples of various lactose concentrations, at a 1:9 (v/v) ratio of inoculum to the culture medium. The experiments were performed in triplicate, with data obtained expressed as mean value ± limits. For statistical analysis, the ANOVA  technique was employed to evaluate the results. Furthermore, data were analyzed to determine the statistical differences between the average measurements of independent groups by performing multiple comparison post hoc t-tests. If a threshold level of significance, p < 0.05, is reached, then differences are considered statistically significant.The inoculum of the The inoculated samples were grown for seven days in an Innova 42R benchtop incubator with a built-in shaker  that allows cultivation at constant temperature and also provides the possibility of sample stirring due to its built-in rotative platform. Cultivation parameters were set at 28 ± 1 °C temperature and 175 rpm, a rotation speed that maintains the microalgae cells in suspension and does not allow adhering to the flask walls without affecting their growth rate.2∙s1 photons. The two illumination programs were proposed in order to determine which is more efficient for contaminant reduction, based on the consensus that the day–night illumination system allows microalgae cells to synthesize, during the high-illumination period, basic molecules, through photosynthesis, using the elements present in the nutrient medium, and in the darkness period, the cells transform the basic molecules in compounds with a higher molecular mass, such as pigments, carbohydrates, proteins, lipids [The illumination programs selected for microalgae growth consisted of both continuous high-light LED illumination and day–night cycle, consisting of 12 h LED illumination and 12 h of darkness, throughout the seven days of cultivation. The LED illumination was supplied by the integrated lighting system of the benchtop incubator, which provided the samples with cool white light with an average photosynthetic active radiation (PAR) of 240 μmol/m, lipids . Microalgae harvesting was done by centrifuging each sample at 7000 rpm for 15 min, using a Rotina 420R Hettich centrifuge. The supernatant was collected separately for analysis of lactose, TN, TP, COD, pH, and salinity. After removing the supernatant, the microalgae biomass was washed with two portions of distilled water to remove residual salts remaining on the surface of the microalgae cells. The wet microalgae biomass was then freeze-dried using a Martin Christ Alpha 1–4 LDpLUS Lyophiliser, and microalgae productivity was determined for each sample.w/v ratio). Extractions were carried out overnight, at 4 °C, under stirring at 150 rpm, followed by removal of supernatant via centrifugation. For each sample, all consecutive effluents from the three successive extractions were reunited and analyzed spectrophotometrically. Absorption spectra were measured between 400 and 800 nm.Dried microalgae biomass samples were grinded thoroughly to achieve a better extraction yield. Weighed samples of dried biomass were submitted to three successive extractions with methanol in order to extract as much of the bioactive compound as possible. For each extraction, the dry biomass to solvent ratio used was 0.03 g to 2 mL  and (2). Concentrations of chlorophyll a and b, as well as carotenoids, were determined using Equations (1)–(3), published in the work of Xiong et al. , based ov/v) chloroform:methanol mixture, at a biomass to solvent ratio of 1:4 (w/v), based on a procedure modified from Folch et al. [w/v/v). The fatty acid methyl esters were isolated from the reaction products via liquid–liquid extraction and phase separations using hexane and a 20% sodium chloride aqueous solution as part of the organic layer, which was then oven dried and analyzed via GC-MS in order to determine their fatty acid profiles.For evaluating the lipid fraction of each microalgae biomass sample, extractions were carried out using a 2:1 . The addition of DCW as a carbon source allows for higher nitrogen and phosphorus reduction rates to be achieved over the control samples, regardless of the illumination system that was used. Furthermore, when factoring the combined stress, organic substrate present in the growth medium in the form of DCW as well as light stress in the form of continuous illumination, both nitrogen and phosphorus reduction rates tend to be higher than those in the day–night illumination cycle, when comparing samples with similar quantities of DCW added. To better understand these results, the microalgae biomass productivity for each sample must be considered.t-tests (n = 5) indicating a significant difference for each sample over the control samples, in both types of illumination cycles. For the day–night cycle, productivity increases gradually, proportional to lactose concentration, whereas, for continuous illumination, productivity values are more than double that for the samples cultivated in the standard growth medium without added DCW. The highest biomass productivity was obtained in the continuous illumination system when the growth medium was modified through the addition of DCW corresponding to a lactose concentration of 2.5 g/L. From With the increase in added DCW to equivalent lactose concentrations of over 5 g/L, biomass productivity was still improved over the control samples. However, this increase in productivity became less significant, especially compared to those obtained for samples with added DCW equivalent to a lactose concentration of 2.5 g/L. Additionally, with this increase in the volume of added DCW, the difference in productivity decreased between samples obtained in continuous light and those obtained in a day–night cycle, most likely as a result of the microalgae cells using the available carbon substrate for growth without relying on photosynthetic metabolic pathways.t-tests (n = 5), showed a significant decrease (p < 0.05) in lipid fractions for samples grown in a medium with added DCW for both types of illumination cycles. Lipid content presented an opposite trend to biomass productivity . The higp > 0.05) between microalgae samples grown with added DCW and control samples, with the exception of sample L7.5, containing added DCW equivalent to a lactose concentration of 7.5 g/L, which showed a significant decrease (p < 0.01). This can be explained by the fact that the microalgae strain uses the lactose and nutrients present in the medium in order to grow and multiply, considering this environment ideal for the growth process. When the microalgae strain is subjected to stress factors, such as the light stress for experiments carried out with continuous illumination, it consumes nutrients present in the growth medium and stores them in the form of lipids and pigments so as to have an emergency build-up [The same can be observed for total fatty acid concentration, with a significant decrease in FAME concentration being observed for the day–night cycle. On the other hand, for continuous illumination, statistical analysis showed no significant differences (build-up .p < 0.05), regardless of the type of illumination, while in the case of carotenoids, there are no significant changes. Increasing the amount of added DCW led to pigment concentrations decreasing visibly in most cases with the amount of DCW added to the growth medium, or in the case of chlorophyll b, showing no significant differences over the control samples. This indicated that at available lactose concentrations higher than 2.5 g/L, the microalgae strain tends to accumulate biomass , oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3). With the presence of various lactose amounts in the growth medium, and considering the illumination system, the distribution shift mainly occurs amongst these four fatty acids. For the control sample (L0), the continuous illumination system aids in increasing the percentage of polyunsaturated fatty acids in the lipid fraction: C18:0 decreases, and C18:1 and C18:2 concentration increases. The shift in fatty acid composition could be a result of the stress factors present during microalgae growth, specifically light stress, which favors the metabolic pathway resulting in the transformation of C18:0 to polyunsaturated fatty acids [The principal fatty acids identified in the lipid fractions extracted from ty acids .With the increase of lactose concentration in the growth medium, the microalgae strain continues to produce higher concentrations of polyunsaturated fatty acids, mainly C18:2 and C18:3. There is also an increase in C16:2 and C16:3 concentrations compared to the day–night cycle of illumination.Nannochloris sp. for the treatment of dairy wastewaters was proven to be efficient, both in the reduction of nutrient content in the medium and also for obtaining microalgae rich in added value compounds such as lipids (up to 25% of dry biomass), with a high proportion of unsaturated fatty acids  and considerable amounts of antioxidants, consisting in chlorophylls, up to 16 mg/g and carotenoids, up to 2.5 mg/g. The results obtained in this study are in accordance with the literature data regarding other strains of microalgae used for wastewater treatment. Plöhn et al. [Scenedesmus quadricauda used for dairy wastewater treatment achieved removal yields of approximately 92% nitrogen and 71% phosphates. The use of microalgae strain n et al.  reportedChlorella vulgaris, with a removal rate of 85% TN, 66% TP, and 81% COD, with maximum biomass productivity of 1.23 g/L after 10 days cultivation; Chlamydomonas polypyrenoideum, with a removal rate of up to 90% TN; Scenedesmus quadricauda, with a removal rate of 86% TN, 90% TP, and 64% Total Organic Carbon, with maximum biomass productivity of 0.47 g/L after 12 days cultivation; Tetraselmis succica, with a removal rate of 45% TN, 42% TP, and 40% Total Organic Carbon, with maximum biomass productivity of 0.56 g/L after 12 days cultivation.Goswami et al.  listed iChlorella vulgaris, with 77% unsaturated fatty acids, Scenedesmus quadricauda, with 75% unsaturated fatty acids, and Tetraselmis succica, with 85% unsaturated fatty acids.Similar research concludes that there is a relationship between nitrogen availability and lipid content in microalgae biomass. When microalgae cells are grown with nitrogen deficiency, the metabolic pathway for carbon fixation changes from protein synthesis to lipid production. It was found that the lower the initial content of nitrogen in the culture medium, the greater the number of lipids in the biomass . The shiThe present research conditions achieved parameter concentration reductions of up to 94% for nitrogen, 82% for phosphorus, and 93% for COD values—for microalgae cultivation under continuous illumination conditions, and up to 90%, 65% and 94%, respectively, for microalgae cultivation under the day–night illumination cycle. The reduction of phosphorus content was visibly lower for day–night illumination cycles. For nitrogen and COD, the threshold percentages of nutrient removal were achieved in both illumination systems and for phosphorus in continuous illumination, as mentioned by the European Directive 91/271/EEC. However, the concentrations for COD, TN, and TP after microalgae cultivation are still higher than those indicated by the Directive, even when the threshold reduction rates are achieved. For a continuous illumination growth system, for example, after cultivation in a growth medium modified by adding DCW equivalent to a lactose concentration of 10 g/L, the average values for TN, TP, and COD were 20, 60, and 900 mg/L, respectively, instead of the required 15, 2, and 125 125 mg/L, respectively (European Directive 91/271/EEC). However, given the initial high organic load, the reduction is significant. Further studies will focus on the potential of using multi-step microalgae-based treatment processes, with effluents not in line with requirements for release into the environment being recirculated and used for growth medium preparation of new microalgae cultivation batches. The nutrient reduction could therefore be reduced to percentages much higher than 90% . For the


This study aims to explore the effect of cochlear implantation on tinnitus perception. A prospective study was conducted on 72 adult hearing-impaired patients to evaluate tinnitus perception before and after cochlear implantation, using standardized tinnitus questionnaires , and tinnitus handicap inventory (THI)). A large variety of demographic and hearing- and implant-related data was collected from patient hospital records to explore possible associations with the implantation effect. The prevalence of tinnitus complaints before implantation was 58.3%. The temporary induction or aggravation of tinnitus immediately after surgery was noted in 20% and 46.7% of patients, respectively. When evaluated 3 months after implantation, 60% of tinnitus patients experienced a clinically significant reduction in their complaints; most of the improvements were experienced immediately after activation of the implant. Only the scores for TFI and THI at baseline were found to be significantly correlated with a reduction in TFI scores after implantation. In 80% of tinnitus patients, the tinnitus remained suppressed for some time after taking off the device. The large subset of patients with residual inhibition supports the involvement of central pathophysiological processes in implantation effects on tinnitus, which are explored in this paper. The prevalence of tinnitus in the general population is reported to be 10–15% ,3,4. HowAs CI is considered the standard treatment for bilateral profound hearing loss , reportsThe limitations of previous research indicate the need to address these bottlenecks: many studies have adopted a retrospective or cross-sectional design using non-validated measuring tools  ,25,26), The objectives of the current study were, first, to investigate the possible effects of CI on tinnitus  in a prospective way, studying a patient group implanted according to classic reimbursement criteria. Second, our goal was to collect quantitative and effect-sensitive data by using the tinnitus functional index (TFI)  for tinnThis prospective, mono-centric study included 72 adult patients receiving a CI at Ghent University Hospital, irrespective of their tinnitus complaint prior to implant. The subjects were primarily implanted for uni- or bilateral severe-to-profound SNHL, whether congenital or acquired. Of these 72 patients, 5 exhibited SSD and 67 exhibited bilateral severe-to-profound SNHL. Their accompanying tinnitus complaints were assessed by presenting tinnitus-specific questionnaires  from Cochlear, Advanced Bionics, Oticon, or MedEL were used.To assess the characteristics and severity/handicap of tinnitus complaints, each patient (irrespective of the presence or absence of a tinnitus complaint) completed a validated Dutch-language version of the tinnitus sample case-history questionnaire (TSCHQ) , the tinPatient age, sex, hearing-loss etiology, onset, laterality, and duration information were collected from some items of the TSCHQ or from patient files and were used for analysis. The type of electrode and its effect on tinnitus, as well as whether a bimodal fitting was used, were also analyzed.Unaided hearing thresholds were collected at baseline and at one month postoperatively to assess residual hearing and hearing preservation, respectively. Aided hearing thresholds for the implanted ear were noted at the 6-month follow-up, together with speech audiometry.χ2 tests were used to compare categorical variables, and Student’s t-test and the Mann–Whitney U test were used for the parametric and nonparametric testing of 2 independent samples of continuous variables, respectively. The correlation between possible associated variables was quantified using the Spearman correlation coefficient. To assess the variability of the THI and TFI scores over time, the nonparametric Friedman test was applied. Post hoc pairwise comparisons were carried out using the Wilcoxon signed-rank test with the Bonferroni correction applied, resulting in a significance level set at p < 0.017.Data processing and statistical analysis were performed with SPSS version 23.0 . For all statistical analyses, a significance level of 0.05 was used, unless otherwise indicated. Descriptive parameters were calculated, and tests of normality  were applied to ensure the correct use of parametric versus nonparametric tests. For inferential statistics, Fisher’s exact and n = 52). The distribution of hearing-loss etiologies is displayed in In total, 72 subjects with a mean age of 55.7 years  were included in the study. Forty subjects were female (55.6%). The average duration of hearing loss was 26.3 years , and most of the study subjects exhibited a post-lingual hearing loss onset , ipsilateral  to the ear considered for implantation, or bilateral , and with a mid-frequency pitch . In total, 12 patients (28.6%) indicated that their tinnitus could be masked by environmental sounds, while 12 out of 34 (35.3%) patients who had a hearing aid before implantation experienced an on/off phenomenon, whereby the tinnitus was suppressed while wearing the hearing aid and immediately returned to baseline after taking off the device. Three of them exhibited some form of RI .The prevalence of tinnitus complaints pre-implantation was 58.3% (42/72 subjects). All the patients with tinnitus suffered from a subjective, non-pulsatile form. The mean duration of the tinnitus was 10.9 years . The mean severity of the tinnitus was significant according to the TFI questionnaire ), and mild when measured by the THI ). The scores are displayed in The baseline characteristics of the CI population are shown in Postoperative data at the 3- and 6-month follow-ups were collected from 30 (71.4%) and 27 (64.1%) tinnitus patients, respectively. Not all tinnitus patients completed the questionnaires at all different time points, usually due to practical considerations.Using a cut-off value of 13 points as a clinically meaningful change in TFI score, 60% (18/30) exhibited a clinically significant tinnitus improvement at 3 months after implantation, compared to pre-implantation, with the subjectively largest improvement recorded close to the time of activation of the implant, approximately 4 weeks after surgery. At the 6-month evaluation, the remitter rate was 70.4% (19/27).2χ(2) = 28.382, p < 0.001]  and between baseline and 6 months post-implantation . No significant differences in TFI scores were seen between 3 and 6 months after implantation at follow-up . Average scores evolved from 41.2 (±29.3) at baseline to 13.0 (±18.4) and 10.3 (±14.6) at 3 and 6 months postoperatively, respectively.Scores for the change-in-tinnitus questionnaire across the three testing time points in the overall group with tinnitus before implantation were evaluated. Only patients with full data sets were included for analysis. There was a statistically significant difference in TFI score over time using the Friedman test [< 0.001] . Post hoχ2(2) = 25.268, p < 0.001], with post hoc testing revealing significant changes between the baseline and the 3-month scores  and between the baseline and the 6-month scores . The THI score again remained stable at follow-ups between 3 and 6 months post-CI . The median THI scores for the 3 evaluation time points were 24, 4, and 1, respectively. The improvements in THI sub-scale scores  were comparable with changes in the global THI score over time.Comparable effects were seen regarding the total THI score: a statistically significant difference over time was found , as also indicated in r = 0.834, p < 0.001 and r = 0.653, p < 0.001, respectively)  . Thus, pn = 19), analyses were also performed to identify possible associated factors. This patient group was compared to the group with stable or suppressed tinnitus at the 3-month follow-up or to the group without any tinnitus at all at baseline and at 3 months post-implantation (n = 35). We observed a significantly shorter duration of hearing loss  and a higher TFI score at baseline  in the subgroup with worsening tinnitus. Additionally, the aggravation/induction group had more post-lingual than pre-lingual hearing loss , as well as more ipsilateral tinnitus and hearing complaints . The groups did not differ in age, sex, tinnitus duration or type, CI electrode configuration, severity of hearing loss, performance with CI (hearing thresholds or speech understanding), or bimodality or hearing preservation.In the subgroup with the (temporary) induction or aggravation of tinnitus post-implantation  in 60% of our population at the 3-month follow-up, which remained stable at the 6-month follow-up. Complete suppression rates of 15–83% or a partial reduction of the tinnitus in 46–95% ,17,33,34Twenty percent of our patients reported the temporary emergence of new tinnitus complaints immediately after the surgery, whereas 46.7% of the tinnitus patients exhibited a temporary aggravation of symptoms. These numbers were higher than we expected, based on previous results. Usually, induction or aggravation rates vary between 0–23% and 4–26%, respectively ,21,22,23However, similar to the observations by Quaranta et al. , our finWe included a large variety of possible demographic and hearing-, implant-, and tinnitus-related factors that might be associated with the suppressive effect of CI on tinnitus. We were only able to show a link between the TFI and THI scores at baseline and an improvement in TFI scores after implantation. Our finding that the severity of complaints at baseline is correlated with the implantation effect confirms previous observations ,17,28, aWith regard to preoperative hearing function, our study was not able to identify any significant variables associated with the effect of implantation on tinnitus, which is in accordance with other studies ,9,10. InIn accordance with the literature, no association was found between a tinnitus implantation effect, on the one hand, and age, sex, duration or severity of hearing loss, tinnitus duration ,10,11,24n = 7), compared to the group without tinnitus induction after CI (n = 43). However, this was again investigated retrospectively and while using only descriptive data. Kompis et al. [As associated factors for the temporary) aggravation or induction of tinnitus after surgery, we found a shorter duration of hearing deprivation, a higher TFI score at baseline, post-lingual onset of hearing loss, and more ipsilateral hearing and tinnitus complaints in this subgroup. This subgroup has not been extensively investigated before, mainly because of its low prevalence. Our finding confirms the results of Pan et al. , who reps et al.  found los et al. . HoweverNumerous hypotheses have been postulated to explain the suppressive effect of CI on tinnitus complaints ,11,37,38The most classic theory is focused on peripheral auditory input changes. By enhancing their hearing capacities through CI, patients can focus on masking sounds and distract their attention toward auditory stimuli other than the perception of tinnitus. This masking mechanism suggests a short-term and transient effect, lasting only as long as the stimulation occurs. This hypothesis may be supported by our finding that most of the suppressive effects occur immediately after the activation of the implant, which is also supported in the literature ,38. The However, increasing research interest is focusing on the possibility of implantation modulating the tinnitus-related activity in the central neuronal system ,39.First, since auditory as well as non-auditory  pathways are involved in tinnitus perception, the improvement in hearing capacity brought by CI may reduce the stress and anxiety experienced by the patient and, thus, improve their tinnitus coping abilities. Consequently, the reporting of a tinnitus handicap in specific questionnaires (TFI and THI) might be significantly reduced, even though the induction of tinnitus might remain unchanged ,11,23.Second, direct stimulation of the cochlear nerve might disrupt its disturbed neuronal synchrony or even central auditory processing in general . MoreoveThis suggests that implantation induces bottom-up processes and dynamic neuroplasticity along central tinnitus-related neuronal networks, as has been shown previously for hearing loss . The exaTo summarize, arguments can be made for masking as well as for central neuroplasticity changes to account for how implantation might influence tinnitus perception. Most likely, both mechanisms play a role to a greater or lesser extent, depending on the individual tinnitus patient. This is possibly supported by the great heterogeneity seen in the clinical expression of tinnitus. However, the high prevalence of RI in our study reflects long-term changes and strengthens the possibility of implantation-induced modifications to central neuronal tinnitus pathophysiology.The added value of our paper in comparison with the available literature is (1) the prospective design, with fixed follow-up times, and (2) the use of TFI as an evaluation method, stated to be sensitive when assessing subtle effects in terms of tinnitus perception ,26. AddiHowever, our large patient group also incorporated many heterogeneous characteristics and included uni- and bilaterally implanted patients, patients with uni- and bilateral hearing loss and/or tinnitus complaints, and those with pre- and post-lingual hearing losses, among others. These factors might mask specific effects in certain subgroups. An analysis of homogeneous subgroups would be useful to draw definitive conclusions. However, this was not feasible in our study due to the resulting small study samples.Also, future research can focus on an exploration of the RI response elicited by electrical versus acoustic stimulation , to gainFor a complaint as subjective as tinnitus, one should always interpret results with caution and bear in mind a possible placebo effect. However, the proportion of responders in our study appears to exceed the classically reported placebo effect . Moreover, we would expect an overreporting of tinnitus complaints in the context of a study where there is more focus on tinnitus and where the subjects are more driven toward it. The reported tinnitus suppression rates could, thus, even be an underestimation of the true effect.The study of effect-associated variables was rather exploratory, focusing on a prospective investigation, outlining the potential effects, the variables, and the observation of residual inhibition, which, in turn, was suggestive of centrally induced changes. However, this implies that the variables described herein cannot be considered independent predictors for the outcome of implantation on tinnitus until this finding is confirmed by subsequent analysis in future studies.Another limitation of the current study is that the first postoperative evaluation of tinnitus complaints took place three months after surgery. Based on the patient reports, most of the tinnitus modulation occurred closer in time to surgery and implant activation; these changes were, thus, noted in a descriptive, retrospective manner. Future research would benefit from the introduction of earlier assessment time points .The prevalence of tinnitus before implantation was 58.3% in our study population. In 60.0% of patients, a significant tinnitus reduction could be obtained at three months after implantation. This effect was correlated with tinnitus severity at baseline. Eighty percent of these patients with tinnitus reduction exhibited temporary, or even long-lasting, suppression after removal of the implant. This indicates the importance of considering sustainable central pathophysiological mechanisms by which CI might influence tinnitus perception compared to immediate effects achieved by enhancing auditory input or the electrical stimulation of the acoustic nerve.