Objective: This study’s objective was to evaluate the effects of pharmacokinetic and pharmacogenetic factors on major bleeding in patients with ACS and non-valvular AF receiving combined antithrombotic therapy consisting of rivaroxaban, clopidogrel, and aspirin as part of dual or triple therapy. Methods: A prospective observational study was conducted in two PCI centers in Moscow, the Russian Federation, from 2017 to 2018. One hundred patients with ACS and AF were enrolled. Prospective follow-ups continued for 12 months. Results: A total of 36 patients experienced bleeding events, with 10 experiencing major bleeding based on the BARC scale and 17 experiencing major bleeding based on the ISTH scale. The following predictors associated with an increased number of major bleeding events were identified: for the ISTH scale, a Css min. of rivaroxaban of >137 pg/mL ) and carriage of the T allelic variant polymorphism ABCB1 rs4148738 ), as well as for the BARC scale ). Conclusions: Measuring residual steady-state rivaroxaban concentrations and determining the carriage of the T allelic variant polymorphism ABCB1 rs4148738 may be applicable to high-risk patients for subsequent antithrombotic therapy modification. Selecting the most suitable antithrombotic regimen can be a complex task, given that approximately 7–15% of patients who have acute coronary syndrome (ACS) also have concomitant atrial fibrillation (AF) .Treatment optimization for patients with AF, ACS, and/or percutaneous coronary intervention (PCI) is being advanced further with the use of randomized clinical trials (RCTs) involving direct oral anticoagulants (DOACs) such as PIO-NEER AF PCI (rivaroxaban) , RE-DUALIn line with current guidelines, patients with ACS and AF undergoing PCI are advised to receive a combination of DOAC and DAT for 1 week. In case of a high risk of recurrent ischemic events, triple therapy may be continued for up to 1 month ,8,9,10. The biotransformation of clopidogrel can be influenced by genetic factors such as the carriage of alleles with a reduced functional activity of CYP2C19 (CYP2C19*2 and *3) and ABCB1 P-glycoprotein polymorphisms ,16,17,18To reduce the risk of bleeding, in addition to influencing modifiable risk factors for hemorrhagic complications, an assessment of the pharmacokinetic parameters of DOACs has been proposed, including polymorphisms in certain genes involved in the absorption, biotransformation, and elimination of DOACs, which can potentially influence the pharmacokinetics of these medications. It has been proven that the metabolism of dabigatran involves the CES1, CES2, and ABCB1 genes . When asA widely utilized tool for evaluating the bleeding risk in patients with AF is the HAS-BLED scale, which aims to quantitatively assess the bleeding risk associated with anticoagulant use. However, despite its extensive use, the applicability of this scoring system in making optimal decisions about antithrombotic therapy for AF patients with indications for combined antithrombotic therapy (ATT) remains uncertain. It is crucial to evaluate which risk prediction tools can consider specific patient characteristics and aid healthcare providers in selecting the most suitable antithrombotic regimen for each case. Therefore, additional factors that could potentially impact bleeding events in this specific patient population must be explored.Study objective: This study’s objective was to evaluate the effect of pharmacokinetic and pharmacogenetic factors on major bleeding according to the BARC and ISTH scales in patients diagnosed with ACS and non-valvular AF receiving combined antithrombotic therapy consisting of rivaroxaban, clopidogrel, and aspirin as part of dual or triple antithrombotic therapy.Study setting and duration. This prospective observational study was conducted at the N.I. Pirogov City Clinical Hospital No. 1 and the S.S. Yudin City Clinical Hospital in Moscow, the Russian Federation, from 1 October 2017 to 30 November 2018.Inclusion and exclusion criteria. The inclusion criteria were as follows: over 18 years of age; presenting with myocardial infarction (MI)/unstable angina (UA) upon current admission; presenting with non-valvular AF confirmed with an ECG; and receiving combined ATT with rivaroxaban as an anticoagulant and clopidogrel only or clopidogrel in combination with aspirin as an antiplatelet agent.The exclusion criteria were as follows: pregnancy and lactation; active internal bleeding; liver cirrhosis with hepatic insufficiency class C according to the Child–Pugh classification; stage 5 chronic kidney disease; HIV infection; alcohol/drug addiction; moderate or severe mitral valve stenosis; mechanical heart valves; severe mental disorders; allergic reactions/drug intolerances; a patient’s unwillingness to participate in this study.This study enrolled patients with MI based on the fourth universal definition of MI. Unstable angina was diagnosed when criteria for myocardial ischemia at rest or upon minimal exertion were present without evidence of myocardial necrosis markers. Initial diagnoses were carried out by the visiting physicians at the hospitals, and final diagnoses were documented in the discharge summary.Follow-up procedures. Following discharge from the hospitals, prospective follow-up continued for 12 months, which included patients with AF and ACS who did not meet the exclusion criteria. The follow-up involved telephone or in-person contact with the patients at the following intervals: one month after inclusion and then at three, six, nine, and twelve months. During the visits, adherence to antiplatelet therapy was assessed using the Morisky–Green questionnaire, and standard laboratory tests were conducted 6 months after discharge.Pharmacological testing and platelet reactivity. Pharmacological testing was also performed. Platelet reactivity was assessed using a portable VerifyNow test system  within 2 h after whole venous blood samples were drawn into 2 mL vacuum tubes containing 3.2% sodium citrate and kept at a temperature range of 2−25 °C. Blood sampling was performed 3–7 days after a patient developed acute coronary syndrome (ACS). The obtained residual platelet reactivity (RPR) is expressed in arbitrary units, PRU (P2Y12 reaction units). The manufacturers recommended using threshold values to interpret the results, with the maximum being PRU = 208 and the minimum being PRU = 95.The quantitative determination of rivaroxaban in blood plasma samples was carried out using the high-performance liquid chromatography method coupled with mass spectrometry according to the established procedure. Blood was collected into vacuum tubes containing 6 mL of lithium heparin 4–5 days after a complete blood count was prescribed before the patient took their next dose of rivaroxaban. The tubes were centrifuged at 15 thousand rpm for 15 min to obtain the plasma. The obtained plasma was aliquoted and stored in Eppendorf tubes at −60 °C until further analysis. This study utilized the following equipment: an Agilent 1200 HPLC System, an Agilent Extend-C18 column, and an Agilent Triple Quad LC/MS 6410 mass spectrometer. The mass spectrum of rivaroxaban was registered using the multiple reaction monitoring (MRM) mode. Threshold values for the minimum steady-state rivaroxaban concentration (Css min.) were defined based on previous research results and EHRA recommendations.Genotyping procedure. For genotyping, venous blood was collected into vacuum tubes containing ethylenediaminetetraacetic acid (EDTA) 3–7 days after acute coronary syndrome (ACS) occurred. The venous blood tubes were frozen at −60 °C until the genotyping procedure was conducted. The identification of polymorphic gene markers’ carriage of ABCB1, CYP3A53, CYP3A422, CYP2C192, CYP2C193, and CYP2C19*17 was carried out using the real-time polymerase chain reaction  method.Statistical processing and analysis. Statistical processing was performed using the Python v3.8 programming language. For quantitative indicators, the nature of the distribution was determined using the Shapiro–Wilk test, including the mean value, standard deviation, median, interquartile range, 95% confidence interval, and minimum and maximum values. The proportional and absolute value counts were determined for categorical and qualitative features. A comparative analysis of normally distributed quantitative features was conducted based on Welch’s t-test (for two groups) or ANOVA (for more than two groups), followed by pairwise group comparisons. When quantitative features were not normally distributed, the analysis was performed using the Mann–Whitney U test (for two groups) or the Kruskal–Wallis test (for more than two groups). A comparative analysis of categorical and qualitative features was carried out using Fisher’s exact test. Regression analysis was used by constructing equations involving one variable to estimate the effects of factors on outcomes. Significant variables were included in a single multiple regression equation to find independent endpoint predictors. Several real significant variables were converted to binary ones, identifying the optimal threshold from the Youden index during the ROC analysis. The significance level for the comparative and regression analyses was 0.05.The study involved 100 patients. The group’s median age was 74 years . Among the participants, 26% experienced MI with ST elevation, 51% had MI without ST elevation, and 23% presented with UA. The prevalent comorbidities in this cohort were hypertension and obesity, as shown in All patients received ATT, including rivaroxaban. Among them, 27 patients (27%) were prescribed DAT consisting of rivaroxaban and clopidogrel, while 73 patients (73%) were prescribed TAT comprising rivaroxaban, clopidogrel, and aspirin.Regarding the specific dosage of rivaroxaban, 20 mg was prescribed to four patients on TAT and three patients on DAT. A dose of 15 mg was administered to 66 patients on TAT and 24 patients on DAT. A dose of 10 mg was recommended solely for patients on TAT and given to three patients. At discharge, patients receiving DAT continued the therapy for 12 months.One month—n = 48;Three months—n = 9;Six months—n = 14;Twelve months—n = 2.The duration of TAT, ranging from 1 to 12 months, varied among the patients, as shown by the following:Thirty-eight patients experienced bleeding during the 12 months of observation. p < 0.0001). By the end of the observation period, 10 patients had major bleeding according to the BARC scale, and 17 patients had major bleeding according to the ISTH scale , RPR (PRU), and the % of platelet inhibition.The distributions of the ABCB1, CYP2C19*2, CYP2C19*17, and CYP3A5*3 genotypes followed the Heidi–Weinberg principle . The CYPp = 0.005), and a higher incidence of stage 3a CKD was observed in this group (p = 0.35). Paradoxically, the proportion of patients with diabetes was higher in the group without bleeding, which was likely due to a statistical error. Apart from these characteristics, both groups were comparable. It is worth noting that there were no differences between the groups concerning HASBLED scores.A comparative analysis of patients with and without bleeding is presented in Considering the indirect impact of rivaroxaban on platelet activity in addition to using the standard PRU 95–208 values for clopidogrel therapy, we explored a more pertinent cut-off point for RPR that could apply to the specific patient population under investigation. To perform logistic regression using ROC analysis, we determined the threshold value of 159 for RPR, with an AUC of 76%, sensitivity of 70.6%, and specificity of 69.9.Subsequently, both univariate and multivariate analyses were conducted on the bleeding scales—BARC and ISTH—with adjustments for gender and age.During the univariate analysis, it was observed that the probability of hemorrhagic complications  increased in the presence of the following factors : carriagAccording to the ISTH scale, the following factors associated with a higher incidence of bleeding events were identified : a Css m-p < 0.004);A higher rivaroxaban concentration and Css min. greater than 137 pg/mL .Carriage of the T allelic variant polymorphism ABCB1 rs4148738 .The influence of the T allelic variant polymorphism ABCB1 rs4148738  and other genetic polymorphisms, did not demonstrate a significant impact on the occurrence rate of major bleeding events.Currently, clinical practice adopts a “bleeding avoidance” strategy to prevent hemorrhagic events. A crucial aspect of this approach involves the development of individualized antiplatelet therapy tactics based on bleeding risk assessment, for which numerous scoring systems have been devised . NeverthThe measurement of DOAC concentrations is becoming an integral part of routine clinical practice, especially for patients undergoing combined ATT. This advancement allows for personalized treatment approaches and the consideration of individual pharmacokinetic profiles. This strategy not only minimizes adverse events but also optimizes therapeutic outcomes, effectively managing the balance between bleeding risks and the prevention of thromboembolic events, a particularly important consideration for patients with AF.While no large-scale study of rivaroxaban plasma concentrations in patients with atrial fibrillation was conducted, we determined the maximum threshold value of Css min. to be 137 ng/mL and the minimum value to be 12 ng/mL. None of the patients in our study exhibited rivaroxaban Css levels below the minimum threshold (<12 ng/mL). Approximately 13% of the patients exceeded the concentration threshold (>137 ng/mL). The median Css min. in our study was 58 , with the highest measured plasma Css min. being 282 ng/mL. These findings are marginally higher than the median rivaroxaban concentrations reported by Al-Aieshy et al., where the minimum concentration was 34 ng/mL . In a stp < 0.004). These findings align with the results of a study conducted on 60 patients receiving rivaroxaban at doses of 20 mg and 15 mg daily, which demonstrated a significant correlation between steady-state rivaroxaban concentrations and bleeding frequency in patients with atrial fibrillation  [A higher concentration of the medication was associated with an increased frequency of major bleeding events, as measured using the ISTH scale  . Moreove = 0.02) .p < 0.018) and the ISTH scales . In several investigations of patients receiving rivaroxaban therapy, ABCB1 gene polymorphisms influenced the plasma concentration of rivaroxaban, but they were not linked to an elevated frequency of major bleeding events [The absorption and elimination of rivaroxaban are primarily influenced by the P-glycoprotein (P-gp) encoded by ABCB1 and BCRP (encoded by the ABCG2 gene) . Accordig events ,33. Neveg events . Subsequg events .Upon completion of the observation period, major bleeding events were observed in 10 patients as per the BARC scale and 17 patients according to the ISTH scale. Such discrepancies in the number of hemorrhagic events can be attributed to the variations in bleeding categorization between the two scoring systems. Similar differences in the incidence of bleeding events, depending on the utilized scoring system, have been described in a sub-analysis of the ENGAGE AF-TIMI 48 trial , which cIn the context of patients with AF undergoing PCI, the significance of TAT becomes notably pronounced. As highlighted by Golwala et al. , the debThe optimal duration and composition of TAT remain subjects of active investigation, as demonstrated by the variability in therapy duration across studies ,4,5. TheA subset of patients in our study received TAT for 3 months (n = 14) and 6 months (n = 2). These decisions were based on the clinical judgment of hospitals and outpatient healthcare providers, considering their own experiences and individual patient characteristics. Prolonged TAT use did not show a significant association with the occurrence of major bleeding events in the multivariate analysis.Study limitations.This study was conducted exclusively at two medical centers in Russia, which may restrict the generalizability of the findings to a broader population. Additionally, the exclusive use of rivaroxaban as an anticoagulant limits the applicability of the results to other DOACs. Moreover, the small sample size of patients should also be recognized as a limitation of this study. To extrapolate the findings to the patient population with ACS and AF, larger-scale studies involving more substantial patient cohorts are required.A higher concentration of rivaroxaban (Css min. > 137 ng/mL) and carriage of the T allelic variant polymorphism ABCB1 rs4148738 are associated with an increased incidence of major bleeding events in patients with ACS and AF who are receiving combined antithrombotic therapy comprising rivaroxaban and clopidogrel. This study’s findings may be applicable to high-risk patients for subsequent therapy modification.


The suitability of the activated biocarbons prepared as the adsorbents of iodine, methylene blue and poly(acrylic acid) from the aqueous solutions was estimated. The sample obtained via chemical activation of the precursor turned out to be much more effective in terms of all the tested pollutants adsorption. Its maximum sorption capacity toward iodine was 1059 mg/g, whereas in relation to methylene blue and poly(acrylic acid) 183.1 mg/g and 207.9 mg/g was achieved, respectively. For both carbonaceous materials, a better fit to the experimental data was achieved with a Langmuir isotherm than a Freundlich one. It has also been shown that the efficiency of organic dye, and especially anionic polymer adsorption from aqueous solutions, is significantly affected by solution pH and temperature of the adsorbate–adsorbent system.Water resources are increasingly degraded due to the discharge of waste generated in municipal, industrial and agricultural areas. Therefore, the search for new materials enabling the effective treatment of drinking water and sewage is currently of great interest. This paper deals with the adsorption of organic and inorganic pollutants on the surface of carbonaceous adsorbents prepared by thermochemical conversion of common pistachio nut shells. The influence of the direct physical activation with CO The presence of organic compounds in water results from various types of natural processes, precipitation, surface runoff, industrial and municipal wastewater ,2,3,4. TDue to the high biological harmfulness of sewage, both municipal and industrial, it must be treated in sewage treatment plants before being discharged into a water reservoir . IndustrIn wastewater treatment processes, mechanical, chemical, biological, mixed methods and disinfection are used . DependiRemoval of impurities from water, including refractive ones, can be successfully carried out using adsorption on the activated carbon surface ,19,20,212 or steam and chemical activation with the use of ZnCl2, K2CO3, KOH, NaOH, NH4NO3, H2SO4 or HCl have turned out to be effective adsorbents of various types of organic and inorganic pollutants. Activated carbons obtained in this way can be successfully used for the removal of NO2 and H2S , MB aq solutionV—the volume of the methylene blue aqueous solution used during the adsorption test [dm3], and samplem—is the mass of activated biocarbon used [g].The adsorbed amount of methylene blue  [mg/g], LK—the Langmuir constant [dm3/mg], ec—the equilibrium concentration of methylene blue [mg/dm3], FK—the Freundlich constant [mg/g (mg/dm3)1/n] and n—the Freundlich parameter (determining the adsorption strength).For the modeling of the equilibrium adsorption isotherms, the Langmuir (Equation (2)) and Freundlich (Equation (3)) equations were used:The poly(acrylic acid)/PAA amount adsorbed was determined using the static method (the decrease in adsorbate concentration before and after the adsorption process). The polymer solution absorbance was measured using the UV–Vis spectrophotometer Carry 100  and by applying PAA complexation reaction with hyamine 1622. As a result, white-colored compound was formed, which absorbs light at a wavelength of 500 nm . The PAAa value of about 4.5 [Poly(acrylic acid)  was characterized by the weight average molecular weights equal to 2000 Da. PAA as a weak polyelectrolyte with an anionic character (pKbout 4.5 ) containbout 4.5 .The conducted studies have shown that waste biomass could be successfully used as a cheap and renewable precursor for the production of activated biocarbons. To a great extent, the physicochemical properties of carbonaceous materials obtained from pistachio nut shells are determined by the variant of the thermochemical conversion applied. Sample activated with orthophosphoric acid has well-developed surface area and porous structure consisting of micropores and small mesopores, whereas analogous product of direct physical activation is characterized by rather poor textural parameters and meso/macroporous structure. Chemically activated biocarbon shows the acidic nature of the surface, whereas for material activated with carbon dioxide, it is alkaline.Adsorption tests have proven that the obtained carbonaceous materials are characterized by very diverse sorption abilities towards inorganic and organic pollutants. The product of chemical activation of pistachio nut shells turned out to be a much more effective adsorbent in relation to all tested pollutants of different chemical structure and molecular size. Its adsorption capacity toward iodine, methylene blue, a synthetic cationic aromatic dye and poly(acrylic acid), an anionic polymer, reached the level of 1059 mg/g, 183.1 mg/g and 207.9 mg/g, respectively. It has been also proven that the efficiency of the organic impurities removal from the aqueous solutions (in particular polymers) is significantly dependent on the pH and the temperature of the adsorbate–adsorbent system.


Although evidence has shown that working memory (WM) can be differentially affected by the multisensory congruency of different visual and auditory stimuli, it remains unclear whether different multisensory congruency about concrete and abstract words could impact further WM retrieval. By manipulating the attention focus toward different matching conditions of visual and auditory word characteristics in a 2-back paradigm, the present study revealed that for the characteristically incongruent condition under the auditory retrieval condition, the response to abstract words was faster than that to concrete words, indicating that auditory abstract words are not affected by visual representation, while auditory concrete words are. Alternatively, for concrete words under the visual retrieval condition, WM retrieval was faster in the characteristically incongruent condition than in the characteristically congruent condition, indicating that visual representation formed by auditory concrete words may interfere with WM retrieval of visual concrete words. The present findings demonstrated that concrete words in multisensory conditions may be too aggressively encoded with other visual representations, which would inadvertently slow WM retrieval. However, abstract words seem to suppress interference better, showing better WM performance than concrete words in the multisensory condition. Working memory (WM) is typically considered a system with limited capacity that can process, store, and monitor information in a short period , which iThe multisensory congruency relationship reflects the tendency to associate seemingly arbitrary features or stimulus dimensions that have a certain relationship under multisensory conditions ,7. PreviThe above studies show that multisensory congruency relationships can affect WM, but the effect of incongruency relationships in multisensory situations is often ignored. Although some studies have revealed that incongruency between targets and irrelevant stimuli can lead to distractions ,11, reseThe effect of multisensory congruency on WM mainly focuses on semantic matching, such as picture and sound  ,14 and wThe present study aimed to confirm and further explore the concept that the multisensory congruency relationship can affect WM performance by adopting an n-back paradigm . We inve2 was determined with reference to a similarly designed three factorial within-subject experiment, and the value was set as 0.25. The two-tailed alpha level was set to 0.05, the power value was set to 0.80, the number of groups was set to 1, and the number of measurements was set to 8. The calculations indicated that a sample size of 30 was needed. Thus, we recruited a total of 30 participants  from campus to participate in the experiment. All participants had normal or corrected-to-normal vision and hearing, were right-handed, were reported to not have mental illness, and had not participated in a similar experiment previously. Individuals were compensated the same amount of money for their participation. After receiving a full explanation of the experiment and potential risks, all participants provided written informed consent in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki), and the study protocol was approved by the Ethics Committee of Soochow University.A statistical power analysis in G*Power version 3.1.9.7 Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany  was perft(46) = 1.998, p > 0.05. Moreover, recent studies have shown that abstract words can elicit more inner properties than concrete words, such as emotions and interoception [t(46) = 8.1, p < 0.001. Finally, an independent sample t test was conducted on the concreteness of the two selected words. The results showed that there was a significant difference in the concreteness of the two types of words, t(46) = 26.83, p < 0.001. Overall, familiarity, concreteness, and emotional arousal were matched between the concrete and abstract words, as shown in The experimental materials included 24 abstract words and 24 concrete words was shown in oception ,29. We chttps://www.neurobs.com/ accessed on 5 March 2022.) under the Windows 10 programming environment. Each visual stimulus was presented on a 27-inch ASUS PG 279 display with a screen resolution of 1920  × 1080  pixels and a refresh rate of 60 Hz on a black background . The central fixation was a white “+” (0.57° × 0.57°); the monitor was located 60 cm away from the participants. Auditory stimuli were presented at a comfortable level through headphones (YINDIAO Q2). The experiment was carried out in a soundproofed, quiet, and dimly lit room where participants used a keyboard (DELL 100) to make key presses, and their responses were recorded.All words were presented visually and audibly in a 2-back task . The sizThe present experiment consisted of a 2 congruency of audiovisual word characteristics (congruent vs. incongruent) × 2 characteristics of target words (concrete vs. abstract) × 2 unisensory retrieval modalities  within-subject design. The experimental designed a 2-back task based on the study by Soveri et al. . Under tIn a gesture to measure the WM of concrete words and abstract words under the unimodal encoding condition and taking it as the baseline, a unimodal encoding condition was also added to the experiment. The 2-back task was administered in 16 blocks, of which the ratio of V:A:AV stimuli was 1:1:1 . The order of the 16 blocks was different for each participant. Each block contained 48 trials, for a total of 768 trials, with a target probability of 37.5%, that is, 18 per block. Because the congruency in the conditions refers to whether the characteristics of the audiovisual presentation of words are congruent rather than whether audiovisual words are the same, the experiment controls congruency under the condition of the audiovisual presentation of congruent word characteristics. The trial ratio of the two types of the same and different audiovisual words was 1:1, and the control of three different types of targets  was 1:1:1. Moreover, to avoid the error caused by the sequence effect on the experimental results, the sequences in each block were pseudorandomly processed three times, and the blocks were randomized so that each participant was faced with a different sequence during the experiment.Each block started with the presentation of a fixation cross (500 ms) and continued with a sequence of synchronized visual/auditory/audiovisual stimulus pairs. Each stimulus had a duration of 500 ms , followed by a blank interval of 2500 ms (ITI), resulting in an interstimulus interval of 3000 ms (SOA). After the end of each block, participants were required to rest for 30 s, and the next block requirement was displayed on the screen. After the experimenter repeated the following instructions and ensured that the participant understood the requirement, the next block could be started after the rest period. Participants were asked to press a button with the right index finger when the current stimulus was the target and press another button with the right middle finger if it was a nontarget. The participant had to respond by pressing the button as quickly and accurately as possible. The mapping between the two response buttons was counterbalanced between participants. Before the formal experiment, each participant was required to complete practice experiments for each condition, the stimulus duration time was the same as that in the formal experiment, and correct/error feedback followed each trial. The formal experiment did not begin until the participants understood and could accurately repeat the experimental requirements. An experimental procedure example was shown in Accurate response rates (ACRs) and reaction times (RTs) were recorded for the sixteen blocks. Accuracy rates were calculated as the percentage of correct responses (correct hits and correct rejections). Trials with no responses or RTs ± 2 SDs beyond the mean RT were not included in the RT analysis . This reProportion correct A 2 congruency of audiovisual word characteristics (congruent and incongruent) × 2 characteristics of target words (concrete and abstract) × 2 unisensory retrieval modalities (V and A) repeated-measures ANOVA was conducted, revealing a significant main effect of unisensory retrieval modalities, F = 18.9, p < 0.001, p = 0.095). In addition, the two-way interaction between the congruency of audiovisual word characteristics and the characteristics of the target word (p > 0.05), between the congruency of audiovisual word characteristics and unisensory retrieval modalities (p > 0.05), and between the characteristics of the target word and unisensory retrieval modalities were all not significant (p > 0.05). Additionally, no three-way interaction was found to be significant (p > 0.05).Reaction time The ACRs for visual and auditory WM retrieval performance reached a ceiling in all encoding patterns (above 90%). A 2 congruency of audiovisual word characteristics (congruent and incongruent) × 2 characteristics of target words (concrete and abstract) × 2 unisensory retrieval modalities (V and A) repeated measures analysis of variance (ANOVA) was conducted and revealed a significant main effect of congruency of audiovisual word characteristics, F = 10.28, p = 0.003, F = 24.29, p < 0.001, F = 10.04, p = 0.004, F = 5.05, p = 0.032, F = 8.03, p = 0.008, F = 10.73, p = 0.003, p = 0.58).To evaluate the effect of the congruency of the characteristics of words with an audiovisual presentation on subsequent unisensory modality WM retrieval under target words with different characteristics, two separate 2 congruencies of audiovisual word characteristics (congruent and incongruent) × 2 unisensory retrieval modalities (V and A) repeated measures ANOVAs were conducted . For theF = 16.84, p < 0.001, p = 0.059). Importantly, the interaction between the congruency of audiovisual word characteristics and unisensory retrieval modalities was significant, F = 15.31, p = 0.001, p < 0.001). These results indicated significantly faster WM retrieval in the visual modality (607 ms) than in the auditory modality (738 ms). There was no significant difference between the visual modality and the auditory modality under the congruent word characteristics condition (p = 0.31). For unisensory retrieval modalities, a post hoc analysis with Bonferroni correction only revealed a significant difference between congruent word characteristics and incongruent word characteristics under the visual modality condition (p = 0.002). This result revealed significantly faster WM retrieval under the incongruent audiovisual word characteristics condition (607 ms) than under the congruent audiovisual word characteristics condition (694 ms). There was no significant difference between congruent word characteristics and incongruent word characteristics under the auditory modality condition (p = 0.31).For the condition in which the target word characteristics were concrete, significant main effects of unisensory retrieval modalities were observed, F = 10.76, p = 0.003, p = 0.75), and there was no significant interaction between the characteristics of the target word and unisensory retrieval modalities (p = 0.48).To evaluate the effect of the characteristics of the target word on subsequent unisensory modality WM retrieval under different congruencies of audiovisual word characteristics, two separate 2 characteristics of the target words (concrete and abstract) × 2 unisensory retrieval modalities (V and A) repeated measures ANOVAs were conducted . For theF = 23.04, p < 0.001, p = 0.58). Notably, the interaction between the characteristics of the target word and unisensory retrieval modalities was significant, F = 9.25, p = 0.005, p = 0.058). This result indicated significantly faster WM retrieval for the abstract word conditions (699 ms) than for the concrete word conditions (738 ms). There was no significant difference between abstract words and concrete words under the visual modality retrieval conditions (p = 0.24). For the characteristics of target word conditions, a post hoc analysis with Bonferroni correction revealed a significant difference between auditory retrieval and visual retrieval under abstract word conditions (p = 0.006 < 0.01), indicating significantly faster WM retrieval in the visual modality (628 ms) than in the auditory modality (699 ms). In addition, another post hoc analysis with Bonferroni correction revealed a significant difference between auditory retrieval and visual retrieval under concrete word conditions (p < 0.001), indicating extremely significantly faster WM retrieval in the visual modality (607 ms) than in the auditory modality (738 ms).For the incongruent audiovisual word characteristics conditions, a significant main effect of unisensory retrieval modalities was observed, The present study aimed to evaluate the effects of characteristically (in)congruent bimodal presentation of words on subsequent unisensory WM retrieval. The presence of lures, along with the fact that all stimuli in the experiment were a perfect bimodal repetition of samples, allowed us to exclude the possibility that participants simply relied on a sense of familiarity to achieve a correct response . AccordiThe results elaborately revealed faster verbal WM retrieval under characteristically incongruent multisensory conditions than under characteristically congruent multisensory conditions, which was closely related to visual and auditory verbal WM processing. The result supports the idea that auditory verbal input has direct access to the phonological store, whereas visually presented materials must be converted into a phonological store . AccordiAdditionally, the results showed that the ignored modality still influenced memory performance, suggesting that the stimuli to be ignored were nevertheless processed at a semantic level, which is consistent with some studies on multisensory WM with the selective attention method . In partIn the present study, we found that under characteristically incongruent conditions, only the auditory WM retrieval of abstract words was faster than that of concrete words. This result indicated that the impact of visual concrete words on the retrieval of auditory abstract words is less than that of visual abstract words on auditory concrete words, which can be comprehended in terms of both verbal working memory and the difference between concrete and abstract words. On the one hand, some neural studies of verbal working memory have also demonstrated that visual letter strings are verbalized as subvocal speech during encoding , and parMoreover, this result supports the idea that selective attention can leave more resources for subsequent higher-order processing and promote the generation of coherent multisensory representations . We hypoFurthermore, the results of the study showed that regardless of whether the characteristics of the target word were concrete or abstract, under the characteristically incongruent condition, visual WM retrieval was faster than auditory WM retrieval. On the one hand, evidence has shown that with concrete targets, there was interference only for words that were semantically similar . For conAdditionally, interestingly, the advantage of visual WM retrieval under the concrete word condition was far greater than that under the abstract word condition. This result once again proves that concrete words seem to invoke the image system and verbal system to promote the generation of robust representations, leading to a stronger visual advantage compared with abstract words . AdditioIn the present study, for the concrete words, the characteristically congruent effect was observed only under the visual retrieval condition. This result indicated that auditory concrete words have a stronger effect on visual WM retrieval of concrete words than auditory abstract words. According to Baddeley’s opinion, auditory verbal information enters directly into the phonological loop . We suggSecond, according to the context availability model, we tentatively suggest that auditory concrete words provide more contextual information from semantic memory compared with auditory abstract words . The larContrary to the result for concrete words under the visual retrieval condition, the characteristically congruent effect was not found under the auditory retrieval condition. The results indicated that, regardless of whether the words were concrete or abstract, the superior robust representation for material presented auditorily relative to that presented visually supports the idea that verbal materials presented auditorily have direct access to the phonological store, whereas visual information must be recoded via articulation . Some stMoreover, only when abstract words were presented in the other sensory modality was visual retrieval faster than auditory retrieval. The dominant role of visual information on WM in multisensory studies seems to be a relatively stable phenomenon ,33. StudHowever, notably, for concrete words under the characteristically congruent condition, there was no significant difference between the visual retrieval condition and auditory retrieval condition. The results indicated that the representation of auditory concrete words has a greater impact on the WM retrieval of visual concrete words, which influences the dominant role of visual stimuli in multisensory encoding. Recent evidence suggests that concrete words are firmly grounded in the sensorimotor system ,49, inclIn summary, the present study investigated the impact of characteristically (in)congruent bimodal presentation of words on WM retrieval. The results showed robust differences between the characteristically congruent condition and incongruent condition; that is, for the characteristically incongruent condition under the auditory retrieval condition, the response to abstract words was faster than that to concrete words. Moreover, for concrete words under the visual retrieval condition, WM retrieval was faster in the characteristically incongruent condition than in the characteristically congruent condition. The present findings provided evidence that concrete words tend to be mistakenly encoded with other visual representations in multisensory conditions, thus slowing WM retrieval. In contrast, the encoding of abstract words seemed to better suppress interference, and, accordingly, WM performance for abstract words was better than that for concrete words.


Papaya fruit has a limited shelf life due to its sensitivity to decay and chilling damage during cold storage. The application of methyl jasmonate (MeJA) is known to reduce the incidence of disease and chilling injury, and to maintain the overall quality of the papaya fruit when stored at low temperature. Consequently, the effects of postharvest MeJA (1 mM) immersion on papaya fruits during low-temperature storage (10 °C ± 2 °C) for 28 days were studied. The experiment revealed that MeJA treatment significantly decreased the papaya fruit’s weight loss, disease incidence, and chilling injury index. Furthermore, the accumulation of malondialdehyde and hydrogen peroxide was markedly lower after the application of MeJA. In addition, MeJA treatment exhibited significantly higher total phenols, ascorbic acid, antioxidant activity, and titratable acidity in contrast to the control. Similarly, MeJA-treated papaya fruits showed higher antioxidant enzymatic activity  with respect to the control fruits. In addition, MeJA reduced the soluble solids content, ripening index, pH, and sugar contents compared to the control fruits. Furthermore, MeJA-treated papaya fruit exhibited higher sensory and organoleptic quality attributes with respect to untreated papaya fruits. These findings suggested that postharvest MeJA application might be a useful approach for attenuating disease incidence and preventing chilling injury by enhancing antioxidant activities along with enhanced overall quality of papaya fruits during low-temperature storage. Carica papaya L.) is a high-quality, delicious berry fruit and is famous for its outstanding nutritional composition and health-promoting properties. However, limited shelf life, massive quality losses, and susceptibility to postharvest diseases during storage are the major challenges facing the papaya industry worldwide [Papaya  is a phytohormone endogenously produced in higher plants and has been long used as a practical approach to maintain the shelf life and quality of many horticultural products because of its numerous physiological activities . The exoExogenous MeJA maintained the nutritional composition of loquats , ChineseExogenous MeJA applications have been widely used to retain and maintain the shelf life and quality of fresh fruits and vegetables. Keeping in mind the above report regarding the beneficial outcomes of MeJA application in attenuation of chilling injury, resistance against biotic and abiotic stress, enhancement of antioxidant activities, and enhancement of shelf life of horticultural commodities, we carried out our studies. However, the previous literature is limited concerning the efficacy of postharvest MeJA application on quality traits and shelf-life preservation of papaya fruits. In the current investigation, evaluations were carried out on the efficacy of MeJA to preserve the postharvest shelf life of papaya fruits under storage at 10 °C. The objective of the study was to assess the effectiveness of MeJA on attenuation of chilling injury, regulation of antioxidants enzymes, and preservation of quality attributes of papaya under low-temperature storage.Carica papaya ‘Red lady’) fruits that were identical in color, size, and shape and free from any apparent disease were picked at the color break stage from a commercial papaya orchard located in Faisalabad, Punjab, Pakistan. In less than two hours, papaya fruits were wrapped in cardboard boxes and delivered to the laboratory. The papaya fruits were again selected in the lab, and those that were devoid of bruises, defects, and cracks were chosen for further tests. The selected fruits were rinsed with tap water, then sterilized for 2 min using a solution of 0.1% sodium hypochlorite, and then air-dried at room temperature (25 ± 2 °C) for nearly 40 min. The surface-sanitized fruits were randomly divided into two lots consisting of 150 papaya fruits each, with one lot being dipped in pure water (control) and the second lot being dipped in 1 mM methyl jasmonate  for 10 min, with each lot having three replicates of 50 fruits per treatment. We kept separate 15 papaya fruits (out of 150 papaya fruits) for weight loss assessment from each treatment. We previously performed a preliminary test on papaya fruit using four different concentrations of MeJA . On the basis of the output of this experiment, the concentration of 1 mM MeJA was kept for future research. All papaya fruits were then air-dried and kept at 10 ± 2 °C and RH of 95% throughout 28 days. Fruit samples (9 fruits per replicate from a lot of remaining 135 fruits) were collected following MeJA treatment at different intervals  across the storage duration to assess the physiological fruit weight loss (PWL), fruit decay, chilling injuries index, accumulation of malondialdehyde and hydrogen peroxide, total phenolics content, ascorbic acid content, the antioxidant activity (DPPH assay), soluble solids contents, titratable acidity, ripening index, pH, and the activity of antioxidant enzyme, as well as sensorial attributes.Papaya  were weighed on the initial day (0 d) and at each storage time interval by using an electronic balance . Fruit PWL was determined by calculating variation between fresh and stored fruit weight and expressed as a percentage.Fruit decay was monitored by counting the rotted fruit number (fruit showing 2–3 spots caused by pathogens) from the total number of fruits at each sampling day of storage time and was expressed as a percentage.Chilling injury in papaya was estimated based on Nian et al.  by usingg. The upper layer was separated and used as a content assay. The collected supernatant (0.5 mL) was stirred with a solution of (0.2 mL of 0.5%) thiobarbituric acid and then heated at 96 °C for 15 min and finally cooled. The mixture was then centrifuged at 15,000× g for 5 min, and the supernatant was monitored at 532 nm, 600 nm, and 450 nm. MDA concentration was estimated as nmol g−1 FW using the following formula.The accumulation of malondialdehyde (MDA) was performed as previously reported by Hodges et al. , with mi2O2) accumulation experiment, a sample of (1 g) papaya flesh tissue was thoroughly homogenized with 5 mL of 1% trichloroacetic acid and centrifuged at 10,000× g for 20 min. Following that, supernatant (0.1 mL) was reacted with potassium iodide (0.1 mL of 1 M) and phosphate extraction buffer , and the solution’s absorbance was measured at 390 nm. The H2O2 concentration was estimated as mmol kg−1 fresh weight [For the hydrogen peroxide  was treated with Folin–Ciocalteu and sodium bicarbonate (3 mL of 700 mM). The absorbance of the solution was measured at 765 nm. The data were given in milligrams of gallic acid equivalent per 100 g of fresh weight (mg 100−1 g FW).The total phenolic content of papaya juice was determined using the method proposed by Hanif et al. . A samplThe radical scavenging activity of DPPH was performed as outlined in Wang et al. . In brieThe papaya ascorbic acid content was determined as outlined in Ruck . A papayThe content of soluble solid was determined with a digital refractometer . For this purpose, papaya fruit was peeled off and crushed in a juice blender. Papaya juice was filtered with Whatman filter paper. A drop from filtered papaya juice was employed to the cleaned prism of the refractometer, and the reading was noted through the scale at room temperature. The prism was properly cleaned with distilled water before and after sample reading. Total soluble solids were represented as °Brix .Papaya fruit’s titratable acidity was assessed as highlighted by Hortwitz . To perfThe ripening index of the papaya fruit was determined by dividing the SSC value by TA, while pH was measured with a digital pH meter.X = standard sugar value used during titration.Y = value of filtrate used during titration.The sugar contents of papaya juice were determined by Fehling’s solution titration method, previously proposed by Hortwitz . Preciseg for 10 min at 4 °C, and the supernatant was separated and used for enzyme assays.The modified technique proposed by Wang et al.  was usedThe SOD enzyme activity was determined as reported in Štajner and Popović . In brie−1 absorbance change was considered as one unit. The protein content was assayed according to the method reported by Bradford [The method of Wang et al.  was usedBradford . BrieflySensory qualities of different papaya fruits were evaluated following the procedure of Lawless and Heymann . Papaya p ≤ 0.05 level was employed to highlight significant differences between applied treatments. Each treatment consisted of three individual biological replications.All statistical analyses of our experiment were executed according to a complete randomized design by using statistic 8.1 software. The data were analyzed by factorial arrangement under a two-way variance assessment (ANOVA) and reported as mean ± standard error (SE). The least significant difference (LSD) test at p < 0.05; p < 0.05). Furthermore, fruits subjected to MeJA treatment were 2.06 times lower in PWL than untreated fruits, as recorded at the end of the storage period . Moreover, MeJA-treated papaya fruits showed a few decay symptoms until day 14 (9-fold lower), and then showed a 3.3-fold lower decay incidence on day 21 of storage than untreated fruits, respectively. At the end of the cold storage period, disease occurrence in MeJA-treated fruits was 2.8 times lower with respect to the untreated fruits  showed 2.11-fold and 1.67-fold lower chilling injury compared to untreated fruits at day 21 and day 28 of storage, respectively  content in papaya fruits gradually increased during postharvest storage. Nevertheless, exogenous MeJA treatment markedly suppressed the increase in malondialdehyde content with respect to control fruits. After day 28 of storage, the concentration of malondialdehyde was noticeably 1.39-fold lower in papaya fruits treated with MeJA than the control fruits (2O2) production rate significantly increased in both MeJA-treated and untreated papaya fruits during the storage period  retained higher levels of ascorbic acid concentration as compared with the untreated group  decreased under storage  decreased ripening index values as compared with untreated papaya fruits. After day 28 of storage, the ripening index was remarkably 0.49-fold lower in MeJA-treated compared to untreated papaya fruits  accumulation is generally associated with oxidative deterioration to the lipid membrane integrity and cold stress. Consequently, the lipid peroxidation level under MDA and H2O2 synthesis is tightly correlated with chilling injury levels and severity [2O2 with chilling injury was also observed during storage  and hydrogen peroxide  indicates the maturity indices of fruits for harvest time and fetches higher prices from the market. Previously, external MeJA application was reported to have ripening-retarding effects on peaches  and sweeCarica papaya L. var Solo 8. Nevertheless, the pattern of change in total sugars exhibited an increasing trend initially followed by a slightly decreasing trend across storage  that combat reactive oxygens species. SOD converts superoxide anion to hydrogen peroxide, and CAT converts hydrogen peroxide to water and oxygen via various metabolic pathways . In the Sensory attributes represent an attraction factor for consumers and depend on sugar and acid composition . In the 2O2) synthesis and accumulation but stimulated the accumulation of phenolics and ascorbic acid content, and led to higher radical scavenging activity values compared to untreated papaya fruits. In addition, postharvest MeJA application clearly enhanced superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) enzyme activity along with acceptable biochemical and sensory quality. Therefore, postharvest MeJA application could be considered as a safe and suitable approach to mitigate disease development.In summary, our findings showed that exogenous MeJA application significantly mitigated chilling injury, conserved higher antioxidant activities, and maintained the quality attributes of papaya. Overall, MeJA-treated fruit exhibited lower physiological weight loss (PWL), disease incidence, and chilling injury than untreated fruits. Moreover, MeJA markedly inhibited malondialdehyde (MDA) and hydrogen peroxide (H


Background and Objectives: The objective of this study was to investigate quantitative changes in cell-free DNA (cfDNA) found in the bloodstream of patients with locally advanced rectal cancer who received neoadjuvant long-course chemoradiation, assuming a change in DNA fragments release during therapeutic stress. Materials and Methods: This was a prospective observational study that involved 49 patients who had three distinct pathologies requiring neoadjuvant chemoradiation: 18 patients with breast cancer, 18 patients with cervical cancer, and 13 patients with rectal cancer. Both breast cancer and cervical cancer patients were used as a control groups. Breast cancer patients were used as a control group as irradiation targeted healthy tissue after the tumor resection (R0), while cervical cancer patients were used as a control group to evaluate the effect of chemoradiation regarding cfDNA in a different setting (squamous cell carcinomas) and a different tumor burden. Rectal cancer patients were the study group, and were prospectively evaluated for a correlation between fragmentation of cfDNA and late response to chemoradiation. Blood samples were collected before the initiation of treatment and after the fifth radiation dose delivery. cfDNA was quantified in peripheral blood and compared with the patients’ clinicopathological characteristics and tumor volume. Conclusion: Thirteen patients with locally advanced rectal cancer (T3/T4/N+/M0) were included in the study, and all of them had their samples analyzed. Eight were male (61.54%) and five were female (38.46%), with an average age of 70.85 years. Most of the patients had cT3 (53.85%) or cT4 (46.15%) tumors, and 92.31% had positive lymph nodes (N2–3). Of the thirteen patients, only six underwent surgery, and one of them achieved a pathological complete response (pCR). The mean size of the tumor was 122.60 mm3 [35.33–662.60 mm3]. No significant correlation was found between cfDNA, tumor volume, and tumor regression grade. cfDNA does not seem to predict response to neoadjuvant chemoradiotherapy and it is not correlated to tumor volume or tumor regression grade. The kit is designed to accurately quantify and determine the size of DNA fragments and smears that fall within the range of 50 to 7000 bp. The red line represents the result prior to radiotherapy, while the blue line represents the outcome after the fifth dose of radiotherapy. We evaluated the area under the curve, which allowed us to estimate the total quantity of DNA fragments between the two collections and determine the ratio and differences between them.2 = 0.1372) and cervical cancer (r2 = 0.064) show a positive regression trend, while breast cancer exhibits a negative regression, with an r2 value of 0.003. It is important to emphasize that the Pearson test did not identify any significant correlations among cfDNA levels (measured both before and on the 5th day of radiotherapy), tumor volumes , and tumor regression grades (TRG), as outlined in In this pilot study, we successfully demonstrated the extraction and standardized quantification of cfDNA from peripheral blood. However, in this specific experiment, a significant correlation was not observed between cfDNA levels and tumor response. This raises questions about the predictive role of cfDNA, which in turn prompts the need for further research and debate concerning the timing and methodology of cfDNA quantification, as its distribution may vary during neoadjuvant therapy.As previously mentioned, a minor quantity of circulating free DNA (cfDNA) is typically detected in healthy individuals. Quantitative studies have shown that in healthy individuals, cfDNA concentrations usually fall within the range of 0–100 ng/mL of blood, with an average of 30 ng/mL, whereas in the blood of cancer patients, cfDNA concentrations exhibit a broader variation, ranging from 0 to 1000 ng/mL, with an average of 180 ng/mL ,18.p = 0.006) [Numerous studies have generated promising findings regarding the utility of circulating cell-free DNA (cfDNA) in monitoring the response to radiochemotherapy in patients with locally advanced rectal cancer. In a study conducted by M. Zitt et al., responders exhibited a reduction in cfDNA to 2.2 ng/mL (with a range of 1.5 to 2.9 ng/mL), while non-responders showed an increase to 5.1 ng/mL (with a range of 3.8 to 10.3 ng/mL) (= 0.006) .p = 0.17; TRG 3, 4 group, p < 0.01) [p = 0.08). This investigation also established a correlation between the total cell-free DNA levels and both the pathological stage and nodal involvement [Furthermore, a separate investigation led by W. Sun et al. revealed a significant decline in the concentration of 400-base pair fragment DNA following chemoradiotherapy, particularly within the group exhibiting a favorable treatment response  . Additioolvement .In our study, we established a control group comprising breast cancer patients undergoing adjuvant radiation therapy (RT). Our hypothesis was that following neoadjuvant therapy and mastectomy, most tumor cells would be eliminated. Adjuvant RT was then administered to irradiate healthy breast tissue with minimal residual tumor cells. Our study highlights a distinction between the breast cancer group, characterized by a limited tumor burden, and the cervical cancer group, which exhibits a more substantial tumor burden and is known for its favorable response to chemoradiation.Before radiation therapy, cfDNA levels in the breast cancer group ranged from 133.90 to 10,241.20 pg/μL, with an average of 1494.92 pg/μL. In the cervical cancer group, the range was from 300.80 to 22,660.00 pg/μL, with an average of 3063.26 pg/μL. On the 5th day post-treatment, cfDNA levels in the breast cancer group ranged from 62.30 to 6196.50 pg/μL, while the cervical cancer group exhibited a wider range and a higher average .The objective of our study was to investigate quantitative variations in circulating free DNA (cfDNA) in patients diagnosed with locally advanced rectal cancer who underwent neoadjuvant long-course chemoradiation. We hypothesize that there may be changes in DNA fragment release during therapeutic stress. To achieve our goal, we established two control groups representing the upper and lower limits of cfDNA levels. Our aim is to differentiate between patients who will respond favorably to neoadjuvant long-course chemoradiation and those who will not. To accomplish this, we worked to establish a correlation between the difference in cfDNA levels before and on the 5th day of radiation treatment and the tumor regression grade (TRG) determined through pathological examination of surgically removed tumor specimens.Unfortunately, among the initial cohort of thirteen patients who initiated neoadjuvant radiochemotherapy, only six patients ultimately underwent surgical resection. Notably, one patient exhibited a complete pathological response classified as Dworak 4, while the remaining patients demonstrated suboptimal responses, with three patients categorized as Dworak 1 and two patients as Dworak 2. Among the seven patients who did not undergo surgery, three were deemed ineligible for surgical intervention due to the presence of metastasis or locally advanced disease, while the unfortunate outcome was observed in four patients who succumbed to causes unrelated to their oncological condition.Truelsen et al. from Elsevier propose that circulating cell-free DNA (cfDNA) can serve as a valuable biomarker and complementary tool to imaging in identifying candidates for a Wait and Watch (W&W) strategy among patients who have achieved a clinical complete response (cCR). They suggest that patients characterized as “cfDNA responders” may have an association with achieving a pathological complete response (pCR). In their investigation, cfDNA samples were collected at baseline, during the midpoint of therapy, and at the conclusion of treatment .In contrast, our study adopted a different approach by collecting cfDNA samples before the initiation of radiation therapy and after the fifth session of radiation. The primary objective was to acquire early results that could assist in distinguishing patients who are unlikely to respond favorably to chemoradiation therapy. This approach sought to identify poor or non-responders at an early stage and to mitigate treatment-related toxicity  and reduIn the subgroup of our study , in one 2 twice daily (BID). By maintaining this control variable, we aimed to establish a positive association between tumor size (GTV), planning target volume (PTV), and cfDNA quantity after the initiation of radiation.Cellular necrosis and apoptosis represent the primary sources of circulating cell-free DNA in plasma following radiation. Another plausible hypothesis involves exploring the correlation between cfDNA, tumor volume and the planning target volume. Several studies have yielded positive findings regarding the relationship between cfDNA levels and tumor size. All of our locally advanced rectal cancer patients underwent long-course radiotherapy (50.4 Gy/28F/5w), either with or without concomitant capecitabine at 850 mg/mRegrettably, despite all patients experiencing a reduction in tumor size following neoadjuvant chemoradiotherapy, no clear relationship between cfDNA and gross tumor volume (GTV), planning target volume (PTV), and tumor regression grade (TRG) was discerned . This laThis study recognizes the need for further research to address the current limitations of cfDNA analysis and enhance its prognostic utility in rectal cancer. It underscores the importance of conducting larger, more comprehensive studies to validate these findings and optimize cfDNA analysis as a prognostic tool for rectal cancer.cfDNA does not seem to predict response to neoadjuvant chemoradiotherapy and it is not correlated to tumor volume or tumor regression grade.


Zinc oxide is a crucial component in rubber products, but its excessive usage can lead to environmental damage. As a result, reducing the amount of zinc oxide in products has become a critical issue that many researchers aim to address. This study employs a wet precipitation method to prepare ZnO particles with different nucleoplasmic materials, resulting in ZnO with a core–shell structure. The prepared ZnO underwent XRD, SEM, and TEM analysis, indicating that some of the ZnO particles were loaded onto the nucleosomal materials. Specifically, ZnO with a silica core–shell structure demonstrated 11.9% higher tensile strength, 17.2% higher elongation at break, and 6.9% higher tear strength compared to the indirect method of ZnO preparation. The core–shell structure of ZnO also helps reduce its application in rubber products, thereby achieving the dual objective of protecting the environment and improving the economic efficiency of rubber products. Zinc oxide is an inorganic filler widely used in the rubber industry, and it has several functions. First, zinc oxide acts as an active agent in rubber to promote the vulcanization reaction, which helps to accelerate the rate of vulcanization and increase the degree of vulcanization, thus improving the physical and mechanical properties of rubber. Second, ZnO is used as a filler of rubber composites intended for products exhibiting increased heat conductivity. Furthermore, zinc oxide acts as an antioxidant that absorbs harmful ultraviolet rays, oxygen, and ozone to avoid their oxidation and the aging of rubber, thus prolonging the service life of rubber ,2,3. In The rubber tire industry represents the largest consumer of zinc oxide, with approximately 50% of total zinc oxide usage being attributed to this industry . Current2 to investigate the effect of SiO2@ZnO core–shell structured nanoparticles on the kinetics of carboxylated nitrile rubber. Yalan L. et al. [With advances in synthesis technology, various methods have been employed to prepare ZnO nanoparticles as a substitute for conventional ZnO. The methods and conditions for preparing ZnO nanoparticles have been extensively investigated and can be broadly categorized as solid-phase, liquid-phase, and gas-phase methods based on the phase state of the reactants ,12,13,14. et al.  used a w. et al.  preparedIn this study, core–shell structured zinc oxide nanoparticles were synthesized via the wet precipitation method, using various materials including calcium carbonate, barium sulfate, silicon dioxide, thermally cracked carbon black, and graphene as the core material. The resulting core–shell structured zinc oxide nanoparticles were characterized in terms of morphology, particle size, and the extent of zinc oxide loading onto the core material. Subsequently, the prepared zinc oxide nanoparticles were incorporated into the formulation of semi-steel radial tire tread rubber, and the effects on the vulcanization characteristics, mechanical properties, friction properties, aging properties, and dynamic thermomechanical properties of the rubber were studied.Zinc chloride used in the preparation was sourced from Shandong Xuanhai Chemical Co., Ltd. , whereas sodium carbonate was obtained from Tianjin Jinhui Pharmaceutical Group Co., Ltd.  The calcium carbonate used in the experiment was provided by Changzhou Calcium Carbonate Co., Ltd.  For the preparation of core–shell structure zinc oxide of barium sulfate, Shandong Qiyi Chemical Technology Co., Ltd.  provided the barium sulfate, and Shandong Bluestar Dongda Chemical Co., Ltd.  provided the silicon dioxide for the core–shell structure zinc oxide of silica. The graphene used in the experiment was sourced from Chinese Academy of Sciences Chengdu Organic Chemistry Co., Ltd. , whereas the thermal cracking carbon black was obtained from Jiangxi Black Cat Carbon Black Co., Ltd. . The indirect method of zinc oxide used in the experiment was provided by Anqiu Hengshan Zinc Industry Co., Ltd. , whereas the other complexes were provided by Guangzhou Chemical Reagent Factory. To prepare the zinc oxide with core–shell structure, the aforementioned raw materials were used, and the experiment was carried out according to the previously described method.3), barium sulfate (BaSO4), silicon dioxide (SiO2), pyrolysis of carbon black (CBp), grapheme oxide (GO), and distilled water were used. To prepare ZnO@CaCO3, 138 g of zinc chloride was dissolved in 330 mL of distilled water. Alternatively, 108 g of sodium carbonate was added to 500 mL of distilled water. The prepared zinc chloride solution was added to the beaker with 81 g of calcium carbonate solids and stirred at 80 °C to make the zinc chloride solution infiltrate the surface of the calcium carbonate solids. Then, the prepared sodium carbonate solution was slowly added to beaker and the solution was continuously stirred at 80 °C in an oil bath. A viscous and honey-like gel was obtained after continuously stirring the solution for 30 min at 80 °C. The solution was washed with water three times and dried for 12 h. Lastly, for the calcination process, a small amount of provided powers were put in an alumina crucible before being placed into a 600 °C furnace. The heating rate was 5 °C/min, and the heating operation was 3 h [To prepare core–shell structured ZnO particles, analytical grade zinc chloride, sodium, carbonate calcium carbonate . The rubber compounds obtained by adding different types of zinc oxide were named R-ZnO, R-ZnO@CaCO3, R-ZnO@BaSO4, R-ZnO@SiO2, R-ZnO@CBp, and R-ZnO@GO.The rubber and auxiliary were mixed in the compactor according to the formulation shown in The crystal structure of the core–shell structured powdered ZnO was determined using a D-MAX2500/PC X-ray diffractometer  with a test range of 10° to 80° and a scanning rate of 5°/min. The morphological characteristics were observed using a scanning electron microscope  with an acceleration voltage of 3 kV. The transmission electron microscope  was used to investigate the microscopic morphology of ZnO with a core–shell structure made from different nucleosomal materials. To test the properties of the prepared core–shell structured ZnO, it was added to a natural rubber formulation. All vulcanization properties were measured using a vulcanometer  at 143 °C. The mechanical properties were tested using an electronic tensile machine  with the sample strips cut into dumbbell-shaped strips with a length of 75 mm, thickness of 2.00 ± 0.03 mm, and working width of 4 mm. The tensile properties were measured at a speed of 500 mm/min. Tear strength was measured using the right-angle tear mode C according to ASTM D624. The thermal oxygen aging chamber was used to age the cut tensile and tear sample strips at 100 °C for 72 h. Abrasion was tested using a DIN abrasion machine  with a pressure of 10 N and a roller speed of 40 r/min. The dynamic mechanical analysis was performed using a dynamic thermomechanical analyzer  with a test temperature range from −60 °C to 80 °C at a frequency of 3 Hz and a ramp rate of 3 °C/min.2, CaCO3, and BaSO4, some different diffraction peaks were detected in the patterns. The XRD patterns of Ca and Ba exhibited characteristic diffraction peaks of calcium carbonate at 23.0°, 29.4°, and 39.3°, and of barium sulfate at 22.7°, 24.8°, 26.8°, and 31.5°, respectively. These observations suggest that the zinc oxide was attached to the surface of calcium carbonate and barium sulfate.The crystallinity patterns and structures of the synthesized samples were analyzed using X-ray diffraction (XRD). 3 powder showed the formation of ZnO nanoparticles, which were spherical in size, uniform, and dense. ZnO was well coated around the nucleosomal material in The morphology of ZnO with different nucleosomal materials is presented in 2 surface, whereas the ZnO morphology on the silica nucleosome material was needle-like. TEM was performed to investigate the ZnO with core–shell structure, as shown in H-ML values than indirect ZnO, which may be due to its lower percentage among the same amount of ZnO, resulting in a slightly lower crosslink density. The core–shell structured zinc oxide exhibits a shorter positive vulcanization time and the fastest vulcanization rate, with a shorter scorch time compared to indirect zinc oxide. This can be attributed to the small particle size, large specific surface area, and severe lack of coordination of the core–shell structured ZnO compared to conventional ZnO, resulting in higher reactivity. In contrast, the vulcanization rate of R-ZnO@CBp and R-ZnO@GO core materials was slower due to the thermal cracking of carbon black and the easy agglomeration of graphene in the rubber, resulting in insufficient reaction between ZnO and the vulcanizing agent.The vulcanization performance of ZnO with different nucleosomal materials in tire tread rubber was evaluated, as shown in The mechanical properties of the rubber compounds are shown in As shown in The loss factors of the materials are shown in The effects of different core–shell materials of ZnO on the aging performance of rubber are shown in This paper proves the core–shell structured zinc oxide was synthesized by a simple wet precipitation method and post-processing approaches without using harmful chemicals. The XRD results showed that the intense and sharp peaks in the ZnO hexagonal were highly crystalline. The SEM and TEM analysis revealed that ZnO nanoparticles (nano-ZnO) are successfully anchored onto carbonate calcium carbonate (CaCO3), barium sulfate (BaSO4), silicon dioxide (SiO2), pyrolysis of carbon black (CBp), and graphene oxide (GO) sheets. The performance of NR/SBR/BR compounds was studied by adding ZnO with different nucleosomes to the formulation of semi-steel radial tire tread rubber. The results showed that the core–shell structured ZnO with low ZnO content possesses a higher vulcanization efficiency and much stronger reinforcement effect on the mechanical performance properties of NR/SBR/BR compounds compared with the indirect method zinc oxide, results of which are positively correlated with the good dispersion of ZnO throughout the NR matrix, the enhanced interfacial interaction between the ZnO and the matrix, and the high vulcanization efficiency of nano-ZnO. For the different core–shell structured zinc oxide materials, the ZnO@silica-based rubber is superior in mechanical and abrasion resistance, and the calcium-carbonate-based core–shell structured zinc oxide has excellent aging resistance. Overall, the performance of core–shell structured zinc oxide product is basically the same as that of indirect zinc oxide products, while the amount of zinc oxide in rubber can be reduced, which can also lead to a reduction in the production cost of rubber products. Accordingly, the core–shell structured ZnO with lower content is very competitive for preparing rubber composites with high performance, and it may be regarded as a substitute of conventional ZnO for application in rubber composites.


Globally, archaeal and bacterial community diversity has been shown to reflect geochemical gradients established by the process of serpentinization. It is unknown if the same is true for microorganisms of the domain Eukarya (eukaryotes). In this study, using 18S rRNA gene amplicon sequencing, we explore the diversity of microbial eukaryotes called protists in sediments of serpentinized fluids in Oman. We demonstrate that protist community composition and diversity correlate significantly with variations in pH, with protist richness being significantly lower in sediments of hyperalkaline fluids. In addition to pH, the availability of CO2 to phototrophic protists, the composition of potential food sources (prokaryotes) for heterotrophic protists and the concentration of O2 for anaerobic protists are factors that likely shape overall protist community composition and diversity along the geochemical gradient. The taxonomy of the protist 18S rRNA gene sequences indicates the presence of protists that are involved in carbon cycling in serpentinized fluids of Oman. Therefore, as we evaluate the applicability of serpentinization for carbon sequestration, the presence and diversity of protists should be considered.In the Samail Ophiolite of Oman, the geological process of serpentinization produces reduced, hydrogen rich, hyperalkaline (pH > 11) fluids. These fluids are generated through water reacting with ultramafic rock from the upper mantle in the subsurface. On Earth’s continents, serpentinized fluids can be expressed at the surface where they can mix with circumneutral surface water and subsequently generate a pH gradient (∼pH 8 to pH > 11) in addition to variations in other chemical parameters such as dissolved CO Serpentogenesis  and manyogenesis . During ogenesis .Archaeal and bacterial community compositions vary along geochemical gradients in serpentinization-hosted ecosystems. For example, a study of an active submarine serpentinization site called Prony Hydrothermal Field, located at the southern end of New Caledonia in the Bay of Prony, demonstrated that community compositions differ by system type, submarine or intertidal . At The 2 and H2. It was demonstrated that the distribution of hydrogenotrophic prokaryote communities corresponds strongly to variations in these geochemical parameters  established by mixing between serpentinized fluid and surrounding surface water in Oman provides an opportunity to assess the influence alkaline pH has on microbial eukaryote diversity. In alkaline environments life can be proton limited, which has consequences for organisms acquiring energy through oxidative phosphorylation . Therefon Turkey . In a stiovolume . It shouHypotrichia. The detection of ciliates at Lost City implies that ciliates may be well suited for hyperalkaline serpentinizing systems on the ocean floor. Serpentinized fluids in Oman provide an opportunity to assess if the same is true in continental serpentinizing systems. Additionally, the pH of serpentinized fluids in Oman varies greatly within the alkaline range, which provides an opportunity to assess the influence pH may have on protist diversity.There is some evidence that protists can live in serpentinization-hosted ecosystems, specifically the deep-sea hydrothermal field, Lost City, located near the Mid-Atlantic Ridge. 2 , and total dissolved Si and sulfide in the field. Samples were collected for lab analysis of major anions , major cations , dissolved inorganic carbon (DIC), trace elements and dissolved gases. The concentrations of major ions, DIC, Si and H2 can be found in 2 gas sample collection and analysis. Combined, the chemical data was used to calculate activities and solute speciation, as well as chemical affinities for mineral dissolution reactions with the geochemical modeling code EQ3  and placed in a −80°C freezer for storage.The protocol used for DNA extraction, modified from methods described in Amplicons of 16S and 18S rRNA genes were sequenced using the Illumina MiSeq v3 2 × 300 platform at the Arizona State University (ASU) Biodesign Institute core facilities. Sequencing library preparation followed a two-step PCR protocol with dual indexing . The firIn the first PCR the template DNA was taken from the same sediment DNA extract for both the 16S rRNA and 18S rRNA gene amplifications. The first PCR thermocycler conditions for the 16S rRNA gene amplification were one denaturation cycle at 94°C for 3 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 48°C for 30 s, and elongation at 72°C for 50 s. A final elongation cycle followed at 72°C for 10 min. The first PCR thermocycler conditions for the 18S rRNA gene amplification were the same for 16S, except for primer annealing at 47°C. The thermocycler conditions for the second PCR were a denaturation cycle at 95°C for 3 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and elongation at 72°C for 50 s. A final elongation cycle followed at 72°C for 10 min. For each amplification PCR reagent mixtures included 15 μL EconoTaq 2x Master Mix, 2.5 μL of each primer (forward and reverse) for a final concentration of 2 μM, 2.5 μL template DNA, and 7.5 μL of sterile, nuclease free water for a final reaction volume of 30 μL. The PCR products were purified at the DNA Shared Resource Facility at ASU using Ampure magnetic beads in combination with a Beckman Biomek NXp robot. Purified products were quantified using Invitrogen Qubit broad range dsDNA fluorescent dye and a Biotek HT1 Plate Reader. After quantification, 25 ng of each amplicon sample, now having a unique barcode and Illumina adapters, were pooled. The pooled sample was then submitted for sequencing.PRJNA919024. Bioinformatic analysis of the FASTQC files was done using the QIIME2-2019.7 (RRID:SCR_021258) program wrapper  files. Sequencing data from this project can be found in the NCBI Sequence Read Archive under the BioProject ID,  wrapper . To gene wrapper .Taxonomic assignment of ASVs was done using a naïve Bayes trained classifier generated from the SILVA 132 99% OTU 16S and 18S databases  and the RRID:SCR_011950) within R version 3.6.0 (RRID:SCR_014212) made by OriginLab Corporation, Northampton, MA, USA. Rarefied ASV frequencies were converted to relative abundances per site to assess variations in composition of 16S and 18S rRNA gene ASVs. Relative abundances were square-root transformed, and Bray-Curtis dissimilarity matrices were generated using the vegan package. To assess drivers of 18S and 16S compositional changes, Mantel correlation analyses with 999,999 permutations were done using the Bray-Curtis dissimilarity matrices and Manhattan dissimilarity of log transformed geochemical parameters. NMDS ordinations of the Bray-Curtis dissimilarity matrices were carried out with 1,000 permutations and two dimensions (k = 2). For both the 18S and 16S ASV NMDS plots, the stress was less than 0.1. The NMDS stress plot for 18S ASVs is shown in RRID:SCR_019129) to assess ASV-driven formation of site clusters as shown in Non-metric multidimensional scaling (NMDS) ordination, Mantel correlation analysis, and analysis of similarity (ANOSIM) were carried out using the vegan package , dissolved H2, dissolved O2 and dissolved inorganic carbon (DIC) are listed in 2 and H2 plotted as function so Si (indicator of mixing) can be found in +2 as chemical modeling shows that aqueous CaCO3 and CO3–2 are the most abundant inorganic carbon species in serpentinized fluids  in two ways. One way is the count of the number of distinct 18S rRNA gene ASVs that occur at each site which we refer to as the observed number of ASVs. Another assessment of alpha diversity employed in this study is phylogenetic diversity calculated using the Faith method . This apr-value > 0.70 and ANOVA p-value > 0.005) as summarized in 2 and the observed number of prokaryote ASVs. Phototrophic protist richness was found to correlate significantly with DIC. Heterotrophic protist richness was found to correlate significantly both with the concentration of O2 and to a greater extent, the observed number of prokaryote ASVs .Using Pearson correlation analysis, protist richness was compared with geochemical and biological parameters that we hypothesize influence protist diversity in sediments of serpentinized fluid. Specifically, we evaluated correlations between overall observed number or protist ASVs and pH, Si (log transformed) and observed number of prokaryote ASVs, all of which are significant , phototrophic protists and dissolved inorganic carbon (DIC) (r-statistic = 0.42) and heterotrophic protists and dissolved O2 (r-statistic = 0.53). Highest Mantel correlations are between protists and the concentration of Si (r-statistic = 0.71), prokaryotes and the concentration of Si (r-statistic = 0.70), heterotrophic protists and prokaryotes (r-statistic = 0.71) and protists and prokaryotes (r-statistic = 0.73).We evaluated variations in community composition, or beta diversity, by conducting Bray-Curtis dissimilarity analysis of the relative abundances of protist 18S rRNA gene ASVs. The relative abundances were square-root transformed. We conducted Mantel correlation analysis between the protist dissimilarity matrix and (1) Manhattan dissimilarity of geochemical parameters and (2) Bray-Curtis dissimilarity of square-root transformed 16S rRNA gene relative abundances. The r-statistic and r-value = −0.86 and ANOVA p-value < 0.005). A significant correlation between Bray-Curtis dissimilarity of square-root transformed protist ASV relative abundances and Manhattan dissimilarity of pH values was also observed , which indicates that variation in protist community composition significantly corresponds to pH.The variation in pH along the mixing gradient observed is in large part due to the process of serpentinization and subsequent mixing between serpentinized fluids and surrounding surface water . In prev+/H+ antiporters or specialized adaptations in ATP synthase . As evident in P < 0.005), which also holds true for presence and absence of ASVs . To investigate whether there is also a distinct transition in protist diversity we carried out NMDS ordination, as shown in Together with the ordination plots and the ANOSIM analysis, the ellipses suggest an overarching community type at sites with <70% serpentinized fluid and another type at sites with >70% serpentinized fluid, with some variability within the two groups. These results, along with observations made in r-value = 0.92 and ANOVA p-value < 0.005. Richness is plotted as a function of log Si in p-value < 0.05  indicates that the most abundant taxon at this site is of the genus, Cyclidium. The site-based dendrogram at the top of We hypothesize that the distinct differences in protist communities between sites with >70% serpentinized fluid and sites with <70% serpentinized fluid can be explained by the physiological differences of protists based on their lifestyle, which dictate the properties of the niche they occupy. To test this hypothesis, we constructed a dendrogram heatmap shown in Cyclidium, and the heterotrophs Ischnamoeba montana, Telaepolella sp. Tib190, and Hypotrichia compose cluster 1 in Chlorophyceae and Spirogyra sp. clade H CC 2012. Taxa within cluster 2 are often the most abundant at sites with compositions that are <70% serpentinized fluid. Cluster 3 is composed of both phototrophs and heterotrophs. Taxa within this cluster are never the most abundant at sites with <70% serpentinized fluid. The same is mainly true for sites with >70% serpentinized fluid as well, except for Nautococcus solutus and Characium saccatum, which are most abundant at sites with 70.1% (site 140110D) and 99.5% (site 140114V) serpentinized fluid, respectively. These taxa are green algae within the clade Chloroplastida. The dominance of Chloroplastida at these sites can be seen in Insights into how the distributions of the most abundant taxa and their composition at each site contribute to the distinct community compositions above and below the 70% serpentinized fluid threshold can be gained from the taxa-based dendrogram and the heatmap coloration. Note that taxa that cluster together in the dendrogram tend to be similarly distributed across sites. As an example, the anaerobic protist genus Chlorophyceae, and ASVs classified as Chlorophyceae are significantly more abundant at sites with <70% serpentinized fluid  and Cylclidum  suggest these sites represent a transition between two community types and the dynamics of these sites, biological, physical and/or chemical, allow for these taxa to co-occur. Site 140117F is located in Shumayt where surrounding surface water and serpentinized fluid are geographically co-located and can mix. Shumayt provides an example of where mixing between fluids in one geographical location may result in a chemical ecotone. Sampling sites from Shumayt are indicated by black boxes in Cylclidium) and phototrophs at site 140117F. Sites 140117F and 140117I may represent systems where intermittent fluid mixing is more likely to occur and therefore may allow two distinct community types to merge, resulting in a chemical ecotone.The dendrogram heatmap illustrates further evidence for a chemical ecotone. As mentioned above, there are two taxa that are phototrophs, a lifestyle that is generally more prevalent at sites with <70% serpentinized fluid, that occur at sites that fall centrally along NMDS1. In the dendrogram heatmap, one of these sites 140110D, is within a site-based cluster that includes site 140117F and site 140114V. Sites within this cluster also have a relatively high abundance of phylotypes classified as Cyclidium. Sites composed of <70% serpentinized fluid are dominated by phototrophs and heterotrophs. Diatoms are particularly distinct in their contribution to the difference in protist community composition above and below 70% serpentinized fluid. Unlike diatoms, species of Chloroplastida appear capable of surviving sites with >70% serpentinized fluid. The two distinct community types that are defined by greater than or less than 70% serpentinized fluid may reflect the physiological properties of the most abundant taxa shown in Overall, the dendrogram heatmap of the most abundant taxa across sites shown in 2, the relative abundances of protists grouped by lifestyle, the richness of protists grouped by lifestyle, the relative abundances of prokaryotes grouped by metabolism type and the richness of prokaryote ASVs. st (25%) and 3rd (75%) quartiles of the data range, and the lines showing the total range of the data.We investigated several geochemical and biological parameters, in addition to pH, that may contribute to the distinct transition in protist community composition and richness at 70% serpentinized fluid. The parameters include dissolved inorganic carbon (DIC), dissolved O2, which is fixed by phototrophs for incorporation into cell mass and respired for energy , or concentration required to reach half the maximum photosynthetic rate, is 100.5 ± 6.6 μM DIC when adapted to high CO2 conditions. When adapted to low CO2 conditions, the half saturation constant is 21.9 ± 3.03 μM DIC  and CO3–2. Inorganic carbon species known to be used by phototrophs, CO2 and HCO3–, are far less abundant at sites where the water consists of >70% serpentinized fluid. While dissolved inorganic carbon is overall low and potentially limiting to phototrophic activity at sites with >70% serpentinized fluid, modeled chemical speciation of inorganic carbon along the gradient shows that CO2 is ∼6 orders of magnitude lower in abundance than HCO3–. While CO2 is more abundant at sites with <70% serpentinized fluid, the thermodynamic activity (≅ concentration) of CO2 is still ∼1 order of magnitude lower than that of HCO3–. Therefore, differences in phototrophic protist community composition may reflect the selection for organisms with carbon concentrating mechanisms (uptake and utilization of HCO3–), particularly at sites with >70% serpentinized fluid. Furthermore, serpentinizing systems may select for protists with novel adaptations that have yet to be observed in nature for taking up and using the more abundant forms of inorganic carbon, CO3–2 and aqueous CaCO3.The correlation between the variation of phototrophic protist community composition and concentration of DIC may be explained by differences in carbon uptake and utilization between protist species. Phototrophic protists carry out photosynthesis with CO2 . Additio for use . This re– to CO2 . As show2-poor conditions  4) can be limiting to growth and survival (2(aq), which is the dominant chemical species of Si in rain and circumneutral surface water, is removed as water infiltrates into the subsurface and reacts with rock, leaving serpentinized fluids relatively depleted in Si  than sites with <70%   significantly correlates with log Si concentration  and richness is significantly higher at sites with <70% serpentinized fluid than at sites with >70% serpentinized fluid composition . For visualization of prokaryote community composition and richness changes along the gradient see We evaluated the composition of potential food sources for heterotrophic protists through 16S rRNA gene sequencing that captures both archaeal and bacterial (prokaryote) diversity and the resulting ASVs were classified using the SILVA database . 16S rRNreducers . A Mantep-value < 0.005, there is a strong correlation between prokaryote and heterotrophic protist community composition dissimilarities. Pearson correlation analysis between the number of prokaryote and heterotrophic protist ASVs shows significant correlation between potential food source richness and heterotrophic protist richness, given the Pearson r-value = 0.85 and ANOVA p-value < 0.005. Overall, these correlations suggest that interactions among bacteria, archaea, and heterotrophic protists shape the microbial ecosystem dynamics along the geochemical gradient. It should be noted that both geochemical selectivity and the influence of predator-prey interactions can induce complex relations among the diversities of heterotrophic protists, bacteria, and archaea. The interactions themselves may be driven in part by the geochemical context, such as availability of oxygen. However, given the great interest in determining geochemical factors that shape bacterial and archaeal communities in serpentinization-hosted ecosystems, the observed correlations highlight the influence protist predation may have on shaping these communities.A correlation between variations in potential food sources and heterotrophic protist community composition is revealed by a Mantel test between Bray-Curtis dissimilarity of square-root transformed relative abundances of prokaryote ASVs and heterotrophic protist dissimilarity. With a Mantel r-statistic of 0.71 and 2 may also influence the diversity of anaerobic protists detected in serpentinized fluids of Oman. Strict anaerobic protists carry out fermentation for energy and facultative protists can switch to fermentation when O2 is limited  of sites where the water composition is <70% serpentinized fluid (n = 7), anaerobic protists are present at less than 1% relative abundance or are absent. In contrast, the relative abundance of anaerobic protists ASVs is >1% at the majority (92%) of sites (n = 12) with >70% serpentinized fluid. While anaerobic protists seem to prefer sites with >70% serpentinized fluid, where the concentration of O2 varies from 8.6 to 203.1 μmol, they are also detected in some of the sites with <70% serpentinized fluid where O2 is nearly at equilibrium with the atmosphere. There is one site where the water is composed of >70% serpentinized fluid that is also nearly in equilibrium with the atmosphere, which is the same outflow site (140115Y) that is relevant to the distribution of the phototroph taxa discussed above. Overall, the distribution pattern suggests that anaerobic protists have a selective advantage at sites where fluids are more influenced by the process of serpentinization and therefore relatively O2 limited.As discussed above, OCyclidium, together with other anaerobic protist species, are known to have methanogen endosymbionts  is most abundant at sites with water that is >70% serpentinized fluid  and not oxidative phosphorylation, thereby bypassing the proton requirement for ATP synthesis. Fermentative protist may therefore have the advantage at sites with >70% serpentinized fluid, which also all have pH > 10.Overall, while not conclusive, the co-occurrence of anaerobic protists and vice versa. Indeed, correlation analysis between dissimilarity of all protist ASVs and dissimilarity of prokaryote ASVs reveals that overall protist community composition strongly and significantly correlates with prokaryote community composition, given the Mantel r-statistic = 0.73 and p-value < 0.005. Richness of protist and prokaryote ASVs are also strongly correlated, indicated by the Pearson r-value = 0.90 and ANOVA p-value < 0.005. While this may be due in part to predator-prey interactions and endosymbiosis, it may also reflect the selective nature of serpentinized fluid. As the contribution from serpentinized fluid increases, community compositions shift and less diversity is supported. Although there are complexities in disentangling the extent to which geochemical processes or biological interactions are driving protist diversity, the serpentinization gradient highlights the possibility that the environment may select for close associations between protists and prokaryotes. Furthermore, the process of serpentinization is thought to be ubiquitous in the solar system and may support life elsewhere such as the icy satellites of Jupiter and Saturn, Europa and Enceladus  can be found below: The lab of GG at Arizona State University provided the supplies and the protocol for assembling the 18S rRNA gene amplicon sequencing library. FD helped AH to assemble the sequencing library. AH conducted the sequencing data processing, analysis, and interpretation with the advisement of FD and GG. AH conducted the geochemical sampling and analysis with the lab of ES at Arizona State University. AH conducted the aqueous chemical modeling with the advisement of ES. AH conducted the interpretation of sequencing and geochemical data and the write up of the results with the advisement of ES, GG, and FD. All authors contributed to the article and approved the submitted version.