The optimization of pretreatment parameters is crucial for obtaining the maximum CNF yield. The response surface methodology was used to design the pretreatment conditions for preparing CNFs. This method consists of four factors: pretreatment time , pretreatment temperature , liquid-to-solid ratio , and PIL content . The predicted CNF yield (Y) followed a quadratic multinomial regression equation represented by Y = 84.43 + 3.59A + 8.22B + 2.22C − 2.13D − 0.85AB + 2.83AC + 5.95AD + 0.43BC − 2.98BD + 4.25CD − 6.04A2 − 18.23B2 − 4.98C2 − 7.39D2. The regression equation exhibited high model fit to the experimental CNF yields as evidenced by a determination coefficient of 0.9764. Results showed that a maximum CNF yield of 86.2% was obtained in the case with the following conditions: pretreatment temperature of 112 °C, pretreatment time of 3.2 h, liquid-to-solid ratio of 83 g g−1, and PIL content of 29%. CNFs with high crystalline index (64.0%) and thermal stability (Tmax = 348 °C) were prepared. This work favors the development of low cost PIL-based pretreatment systems for the clean production of CNFs.Pretreatments with aqueous protic ionic liquid (PIL)–ethanolamine bis ([MEA][(HOA)(H 2OA)]), combined with ultrasonic disintegration, were employed in cellulose nanofibril (CNF) production from pulp fibers.Pretreatments with aqueous protic ionic liquid (PIL)–ethanolamine bis ([MEA][(HOA)(H Cellulose is easily obtainable, inexpensive, nonpolluting, and nontoxic; hence, it exhibits the possibility of addressing many problems, including energy shortage, resource scarcity, and environmental pollution.2 Given its highly linear internal structure, cellulose is strongly influenced by inter- and intramolecular hydrogen bonds; structurally, the coexistence of supramolecular structures in the crystalline and noncrystalline regions encompasses a large number of reactive groups, such as hydroxyl groups, reducing the accessibility of cellulose to reactions and hindering its further industrial application.3 The crystalline region of cellulose has a dense structure and stable properties compared with its noncrystalline region. By physically or chemically removing some of the noncrystalline regions of cellulose while retaining its crystalline regions, nanocellulose can be isolated from micron-scale cellulose materials, considerably expanding the industrial applications of cellulose.4Cellulose is a biopolymer composed of β-As a special class of nanocellulose, cellulose nanofibrils (CNFs) have a diameter of 5–60 nm, a length of several micrometers, and a high length-to-diameter ratio. Their raw material is natural cellulose, and thus, CNFs have renewable and degradable properties. CNFs also exhibit the advantages of nanomaterials, such as high specific surface area, high mechanical strength, low thermal expansion coefficient, chemical stability, ultrafineness, and ultralightness. CNFs have a wide range of applications in many fields, such as in construction, coatings, paper, automobile, food and medicine.5 ILs exhibit the advantages of stable physical and chemical properties, low vapor pressure, and structure designability. Compared with the traditional pretreatment, IL pretreatment demonstrates advantages in solvent recoverability and morphological control of CNFs.6At present, the primary production method for CNFs is as follows: cellulose materials are first chemically pretreated, and then treated through mechanical processing. The traditional CNF preparation method suffers from high energy consumption, low yield, environmental pollution, and other problems because of the use of high concentrations of acid or other chemical reagents. Considering these problems, the use of ionic liquids (ILs), which are environment-friendly solvents, as pretreatment solvents/swelling agents/catalysts in preparing CNFs has been explored.et al.7 prepared CNFs by integrating ultrafine milling into a two-step process with SO2-switched diazabicyclic monoethanolamine IL (SIL) pretreatment. The energy consumption of SIL pretreatment was reduced by 50% compared with that of the conventional process. Zhao et al.8 used acidic ILs-1-butyl-3-methylimidazole hydrogen sulfate ([C4C1im]HSO4)-catalyzed organic solvent pretreatment and ultrasonic disintegration two-step method to extract CNFs from wood flour. The yield, morphology, crystallinity, chemical structure, and thermal stability of CNFs were investigated. The results showed that the yield of CNFs was 41.82% after pretreatment with 1,4-butanediol aqueous solution-[C4C1im]HSO4, and their thermal stability and film-forming properties were superior to those of the products obtained using the concentrated acid hydrolysis method. Although the pretreatment of ILs provides a promising development in the preparation of CNFs, its high price poses a limitation to the practical application of ILs.Berglund 9 During the synthesis of PILs, the addition of an excess amount of acid (relative to a base) results in the formation of dimerization, oligomeric anions, and even anionic clusters, which increase the acidity of PILs. PILs with oligomeric anions have been used in catalysis and biomass pretreatment.10 On the basis of 1-methylimidazole and the anionic cluster [(HSO4)(H2SO4)x] , Paredes et al.11 synthesized a series of PILs for the acid hydrolysis of cellulosic materials to produce cellulose nanocrystals . CNC yields were 60–73% at 40 °C and 2–3 h. Treatment conditions were milder, and the thermal stability of the prepared CNC was better compared with those of sulfuric acid and other PIL treatments in the literature. However, this PIL suffers from the high toxicity of imidazole cation and the high cost of raw materials.Inexpensive proton-based ILs (PILs), which are suitable for industrial applications, can be prepared using nitrogen-containing bases and varying amounts of Brønsted acid. The acidity of PILs is modulated by varying the amount of acid added to prepare a strongly acidic system.2OA)]−) followed by ultrasonic disintegration was investigated. The cation type, i.e., [ethanolamine ([MEA]+), diethanolamine ([DEA]+), and triethanolamine ([TEA]+)], and water addition considerably influenced CNF yields. Under the pretreatment conditions of 110 °C and 3 h, CNF yields with different PIL-based systems exhibited the following order: [MEA][(HOA)(H2OA)] > [DEA][(HOA)(H2OA)] > [TEA][(HOA)(H2OA)]. Overall, CNF yields with PIL–water pretreatments were higher than that with oxalic acid–water pretreatments. PIL–water is an efficient pretreatment system due to its inhibitory actions on excessive cellulose hydrolysis. Thus, the PIL, ([MEA][(HOA)(H2OA)]), was used to pretreat cellulose materials for producing CNFs in the current study. Compared with imidazolium-based ILs, the advantages of [MEA][(HOA)(H2OA)] are lower cost and toxicity.In the preliminary experiment, the preparation of CNFs with the pretreatment that used alkanolamine-based PILs with dimeric oxalic acid anions ([(HOA)(H2OA)] content. Thus, the optimization of the pretreatment parameters for CNF production is crucial for obtaining maximum yield,12 improving the feasibility of commercial applications for the produced CNFs.Pretreatment plays important roles in producing nanofibrils and influencing the properties of the prepared CNFs. The preparation of CNFs is considerably influenced by various pretreatment parameters, such as liquid-to-solid ratio, temperature, time, and [MEA]-based systems for CNF production under the pre-optimized conditions of ultrasonic disintegration. RSM was used to optimize the effect of pretreatment parameters on the yield of CNFs. The most significant parameter was identified in the optimization. The physicochemical properties of the prepared CNFs were analyzed via X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric (TG) analysis. To the best of our knowledge, no report has yet been made about the optimization of PIL pretreatment based on dimeric oxalic acid anions for CNF production. This work is beneficial for the commercial application of CNFs with the use of low-cost PILs.Ultrasonic disintegration considerably influences CNF production and pretreatments. However, considering the complexity of optimization to all affecting factors, this study focused on pretreatments that used [MEA]. Then, 63.3 g (about 0.5 mol) of oxalic acid dihydrate was dissolved in an appropriate amount of ethanol with stirring in a 500 mL round bottom flask. Thereafter, 15.3 g (about 0.25 mol) of ethanolamine was added dropwise with vigorous stirring; dropwise addition was performed in an ice water bath.15 After dropwise addition, the mixture was continuously stirred at room temperature (25 ± 1 °C) until a homogeneous and stable liquid phase was formed, followed by spin-drying at 35 °C to remove ethanol. The synthesized IL was placed in a vacuum oven (with built-in phosphorus pentoxide) at 60 °C for 48 h to remove water. Deionized water was added to the synthesized IL, and aqueous solutions of [MEA][(HOA)(H2OA)] (hereafter abbreviated as PIL–water) with different mass concentrations (10–50%) were prepared under magnetic stirring conditions.IL was prepared by reacting ethanolamine and oxalic acid dihydrate as the starting material in a molar ratio of 1 : 2, The resulting product was called [MEA] aqueous solution system exhibits good pretreatment capability. The CNFs are filamentous in the TEM images, and lengths are difficult to measure due to mutual entanglement. However, all values are known to reach the micron level, indicating that the lengths of the prepared CNFs are relatively small. This finding indicates that the prepared CNFs have relatively large length and diameter, with a length-to-diameter ratio of 104.7. This result is comparable with the isolation of carboxylated nanocellulose length-to-diameter ratio from skimmed cotton by using oxalic acid as determined by Lin et al.26The SEM and TEM images of the CNFs are shown in 3.5.2Cellulose crystal structure is one of the important parameters for determining the physical properties of CNFs. The XRD spectra of CNF and pulp fibers are shown in 3.5.3−1 are attributed to the stretching vibration of –OH.27 The absorption peaks near 1636 cm−1 are attributed to H–O–H planar bending vibration, which is due to the hygroscopic property of cellulose.28 The peaks at 1440–1400 cm−1 are attributed to –CH2 vibration and C–H stretching, which are correlated with the crystallinity of the fiber material. The peaks near 2880 cm−1 are attributed to C–H.29 The peaks near 1165 cm−1 are attributed to cellulose C–O–C vibrations at the glycosidic linkage.30 The peaks near 890 cm−1 are attributed to the vibration of C1.31 The above characteristic peaks are considered typical absorption peaks of cellulose, indicating that PIL–water did not introduce any new functional groups during the pretreatment of pulp fibers, and no derivatization reaction occurred.The FTIR spectra of the pulp fibers and CNFs are shown in 3.5.432 The 370–600 region mass change rate decreased. This stage involves carbon residue decomposition into gas products. As shown in Tmax of CNFs (348.4 °C) was slightly lower than that of pulp fibers (359.2 °C). This phenomenon might be caused by the breakage of cellulose chains, the smaller size of CNFs, increased surface area, and increased heat transfer rate during pretreatment and sonication. Moreover, the large amount of free hydroxyl groups on the surface of CNFs accelerated the decomposition of cellulose glycosyl units.The TG curves of pulp fibers and CNFs are depicted in 42OA)]–water, followed by ultrasonic disintegration. The operating parameters, including pretreatment time, pretreatment temperature, liquid-to-solid ratio, and PIL content, were considered the major influencing factors of CNF yield. The designed response surface experiments confirmed that the four single factors exerted significant effects on the response values. The interaction of IL content with pretreatment time, pretreatment temperature, and liquid-to-solid ratio was significant. The CNF yield of 85.0% produced by the fitted optimal pretreatment conditions was nearly identical to the actual values of 86.2%, indicating that RSM can provide a theoretical basis for optimizing CNF yield. Compared with pulp fibers, CNFs with higher CrI, length-to-diameter ratio, and thermal stability were prepared under the optimized conditions. This work revealed that pretreatment with aqueous [MEA][(HOA)(H2OA)] solutions exhibits considerable potential for high CNF yield in terms of low cost and clean production.CNFs were successfully prepared from pulp fibers with pretreatments that used [MEA][(HOA)(HXincheng Peng: conceptualization, methodology, software, formal analysis, writing – original draft. Deqin Zhu: methodology, software, formal analysis, writing – original draft. Jingjing Liu: formal analysis, investigation. Ligang Wei: supervision, writing – review & editing. Na Liu: formal analysis, investigation. Li Wei: formal analysis, investigation. Guolin Shao: writing – review & editing. Qingda An: funding acquisition, supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.RA-013-D3RA06930C-s001


More specifically, the b‐TiO2 treated with sodium borohydride produced excessive oxygen vacancies resulting in oxygen vacancy band that narrowed the b‐TiO2 band gap, and the small band gap led to NIR‐II region wavelength (1064 nm) absorbance. Furthermore, the combination of defect energy level trapping carrier recombination heat generation and conjugate heat generation mechanism, significantly improved the photothermal performance of the PTT agent based on b‐TiO2. The photothermal properties characterization indicated that the proposed dual‐PTT agent possesses excellent photothermal performance and ultra‐high photoconversion efficiency of 64.9% under 1064 nm laser irradiation, which can completely kill esophageal squamous cells. Meanwhile, Gd2O3 nanoparticles, an excellent magnetic resonance imaging (MRI) agent, were introduced into the nanosystem with similar dotted core–shell structure to enable the nanosystem achieve real‐time MRI‐monitored cancer therapeutic performance. We believe that this integrated nanotherapeutic system can not only solve the application of PTT in the NIR‐II region, but also provide certain theoretical guidance for the clinical diagnosis and treatment of esophageal cancer.Photothermal therapy (PTT), as an important noninvasive and effective tumor treatment method, has been extensively developed into a powerful cancer therapeutic technique. Nevertheless, the low photothermal conversion efficiency and the limited tissue penetration of typical photothermal therapeutic agents in the first near‐infrared (NIR‐I) region (700–950 nm) are still the major barriers for further clinical application. Here, we proposed an organic/inorganic dual‐PTT agent of synergistic property driven by polydopamine‐modified black‐titanium dioxide  agent application. Here, an efficient organic/inorganic dual‐PTT agent guided by MRI was proposed. It is proved the oxygen vacancy band narrowed its band gap that led to NIR‐II region absorb. Furthermore, the defect energy level trapping carrier and conjugate synergistic heat generation result in high photoconversion efficiency. However, TiO2 can only absorb energy in the ultraviolet region (272–390 nm) owing to its large band gap (∼3.0 eV). Ultraviolet light lacks deep tissue penetration and poses side effects to normal tissues, thus greatly limiting the application of TiO2 in the clinic. Therefore, many efforts have been made to further expand its absorption range from the ultraviolet to the near‐infrared (NIR) region, such as hydrogenation reduction, doping, and other methods  to optimize its electronic energy level structure, shorten the forbidden band width, and improve optical performance. For instance, Ren et al. first presented a black hydrogenated TiO2 (H‐TiO2), where white TiO2 reacted with hydrogen plasma at a high‐power density introducing Ti3+ and oxygen defect that turned it into black H‐TiO2. Consequently, the black H‐TiO2 exhibited NIR light‐triggered photothermal performance with excellent photothermal conversion for tumor therapy, which was referred to its dramatically enhanced nonradiative recombination. In order to achieve diagnostic and therapeutic multi‐functions, Wang et al. synthesized a Fe@γ‐Fe2O3@H‐TiO2 nanocomposite by one‐step hydrogen reduction that showed outstanding photoconversion efficiency under 808 nm laser irradiation resulting from the narrow band gap (1.971 eV) of H‐TiO2 and multiple circuit loops for electron transitions between H‐TiO2 and γ‐Fe2O3. Notably, the nanocomposite possessed magnetic resonance imaging, PTT, and tumor targeting functions. It is noteworthy that the photothermal therapeutic agents based on black TiO2 (b‐TiO2) nanocomposites are mainly applied in the first NIR (NIR‐I) region (700–950 nm), which requires a high laser density (2 W cm−2) due to the limitation of light absorption and scattering by tissues. However, this laser density exceeds the maximum allowable skin exposure set by the American National Standards Institute , and has potential damages to the surrounding normal tissues. Hence, the PTT therapeutic efficacy and safety of b‐TiO2 need to be further improved.Photothermal therapy (PTT), as a novel cancer treatment method with high selectivity, minimal invasiveness, and strong operability, has been extensively developed into a powerful cancer therapeutic technique over the past decades. In particular, photothermal therapeutic agents can locally generate hyperthermia to ablate tumor cells in the diseased area when activated by a special wavelength.1 Tita2‐based photothermal therapeutic agents responsive in the second NIR (NIR‐II) region (1000–1500 nm) has garnered significant interest, due to long wavelength light with higher maximum allowable skin exposure  and deeper tissue penetration depth because of less tissue scattering. For example, Guo et al. used mild hydrogenation with NaBH4 method to synthesize b‐TiO2 with abundant oxygen vacancies on its surface that display high photoconversion efficiency of 29.44% in the NIR‐II region, which exhibit precise potential for photoacoustic image‐guided tumor‐targeted PTT. Han et al. constructed core/shell‐structured b‐TiO2‐x nanocomposites via aluminum‐reduction method. The results showed that oxygen‐deficient TiO2–x layer can also enhance the photothermal conversion efficiency (39.8%) of b‐TiO2 nanoparticles under 1064 nm laser for photothermal hyperthermia. These studies successfully developed the photothermal agents based on b‐TiO2 to be excitable in the NIR‐II region and effectively caused the death of cancer cells under photothermal treatment, which solved the drawback of shallow tissue penetration and low maximum permissible exposure. However, the existing b‐TiO2‐based photothermal therapeutic agents generally show a very broad absorption and low photothermal conversion efficiencies. Consequently, it is urgent to develop b‐TiO2‐based photothermal therapeutic agents with excellent photoconversion efficiency in the NIR‐II region, which can alter more light energy to thermal energy for efficient PTT.Recently, the development of b‐TiOattering.10 For PDA nanoparticles exhibit excellent photothermal conversion properties and are emerging as a novel PTT agent for tumor. Specifically, the polymer contains a π‐π conjugated structure, similar to many aromatics and conjugated molecules, which is conducive to the migration of free electrons and carriers, so that it has broad‐spectrum absorption of light in the ultraviolet‐near infrared region. Based on this, we believe that combining PDA with b‐TiO2 can not only improve the biocompatibility of the photothermal therapeutic agent based on b‐TiO2 nanoparticles, but also significantly improve its photo‐absorption performance and photothermal conversion capacity in the NIR‐II region through the synergistic photothermal effect of b‐TiO2 and PDA, to achieve a highly efficient photothermal therapeutic agent for tumors. Meanwhile, the single diagnosis or treatment strategies have been difficult to achieve the clinical requirements. The combination of various diagnosis and therapy technologies to build a diagnosis and treatment platform is the key research direction currently. Therefore, gadolinium oxide (Gd2O3) with appreciable biocompatibility and magnetic resonance (MR) imaging performance can be introduced into b‐TiO2 that realizes the MR image‐guided therapy for cancer.Dopamine, a neurotransmitter in the human brain, has good biocompatibility and easily oxidation and self‐aggregated to form polydopamine (PDA) under alkaline conditions (pH = 8.5).14 PDA2 was prepared by one‐step hydrogenation reduction method, while Gd2O3/b‐TiO2 composite nanoparticles with dotted core‐shell were prepared by polyol method. On this basis, Gd2O3/b‐TiO2@PDA nanoprobes with core‐shell structure were obtained by the oxidative self‐aggregation of dopamine hydrochloride. The nanoprobes not only extended the light absorption of b‐TiO2 to 1064 nm that solved the shallow tissue penetration depth in NIR‐I region, but also provided extremely high photoconversion efficiency of 64.9% at the laser irradiation density of 1 W cm−2 that evaded the irradiation laser density exceeding maximum allowable skin exposure. In addition, the constructed nanoprobes were applied to esophageal squamous cell carcinoma cancer model, and the results showed that the nanoprobes could offer high‐signal MR imaging on the tumor region and had a significant therapeutic effect on the tumor cells. Further, the application of these multifunctional nanoprobes not only reflects the advantages of integrated diagnosis and treatment technology at the NIR‐II region, but also promotes the research on the diagnosis and treatment technology of esophageal squamous cell carcinoma.In this work, b‐TiO22.12O3/b‐TiO2@PDA nanoparticles is shown in Scheme 2 nanoparticles, on this basis, Gd2O3/b‐TiO2 nanoparticles were prepared by a polyol method. In order to increase the biocompatibility of the materials, Gd2O3/b‐TiO2@PDA nanoparticles were obtained by oxidative self‐aggregation of dopamine hydrochloride. As shown in HRTEM image  of anatase phase TiO2 , the weak peaks belonged to the rutile phase TiO2 , indicating the anatase crystal structures was the majority phase in b‐TiO2 and Gd2O3/b‐TiO2. In addition, there were no obvious characteristic peaks in XRD spectrum, representing that the Gd2O3 existed in Gd2O3/b‐TiO2 nanoparticles in the amorphous form because of the small size. The Gd (3d) XPS spectra of the Gd2O3/b‐TiO2 showed that the characteristic peaks of Gd 3d3/2 and 3d5/2 at 1219.78 and 1187.42 eV, respectively, corresponding to the different binding energies with different spin orbits of Gd in Gd2O3  in PDA, respectively  with a poor crystal form and the atomic arrangement was in a highly disordered state , Raman spectroscopy, and XPS. Specifically, the GPA result indicated that the P25 disorder degree was approximately zero and the image color was uniform, indicating that the crystal form of the particles was complete and the atomic arrangement was highly ordered Figure . Howevere Figure . The ress Figure , mainly igure 3+.23 In s Figure , which c2, it can be obtained that the band gap widths of P25 and b‐TiO2 were 2.91 and 1.19 eV, respectively  and concentration of Gd, which represented the efficiency of the nanoprobes as contrast agents. As shown in Figures 1 values of Gd2O3/b‐TiO2 and MAGNEVIST were 7.05 and 4.81 mM−1 s−1, respectively. The r1 value of Gd2O3/b‐TiO2 was much higher than that of MAGNEVIST, indicating that Gd2O3/b‐TiO2 had a stronger shortening T1 effect and more suited for positive MRI contrast agents. The r2/r1 value of Gd2O3/b‐TiO2 was 1.61, indicating that the Gd2O3/b‐TiO2 nanoparticles had excellent T1 MR imaging performance. The T1‐weighted MR imaging showed that the imaging signal was strongest when the concentration of Gd at 0.5 mM  contrast compared with higher concentration of Gd in MR images. Compared with the commercial MAGNEVIST, the nanoprobes had a stronger imaging signal at low concentrations, which indicated the nanoprobes had higher safety. Meanwhile, the MR imaging signal of Gd2O3/b‐TiO2@PDA was highly consistent with that of the Gd2O3/b‐TiO2, indicating that the PDA encapsulation does not affect the imaging performance  was directly observed using a three‐dimension (3D) soft X‐ray microscopy. Due to the large size of a single KYSE‐150 cell, Video 2.52O3/b‐TiO2@PDA nanoprobes in vitro, the cell survival rate was evaluated on KYSE‐150 cells under corresponding conditions. As shown in Figure 2O3/b‐TiO2@PDA  were collected after treatment for the H &E staining. As shown in Figure 2O3/b‐TiO2@PDA had no obvious damage to the tissue, indicating the nanoprobes had appreciable biocompatibility in vivo. To further analyze the safety of the nanoprobes, the blood was collected after treatment for hematological analysis and blood biochemical analysis. As the results shown in Figure 2O3/b‐TiO2@PDA and saline had no significant change in the indicators of hematological and blood biochemical analysis, revealing that the nanoprobes having good biocompatibility ensure its possibility of clinical application.The biocompatibility of the Gd2.71‐weighted MR imaging was performed on the KYSE‐150 tumor‐bearing nude mice by using a 3.0 T MAGNETOM Prisma MR device. Figure 2O3/b‐TiO2@PDA nanoprobes possessed excellent T1 enhanced MR imaging performance in vivo. The thermal imaging of mice treated with the nanoprobes and saline was significantly different, indicating the nanoprobes exhibited considerable photothermal performance and combined with MR imaging can provide a dual‐modal imaging system  2019‐0005).Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.


Varroa mites) and pathogens . We also correlated the expression of genes linked to immune functioning, oxidative stress, and buffering against temperature and air quality stressors. High daily temperatures are associated with poorer air quality, and they are both associated with lowered immune system functions and increased oxidative stress protection against pests and diseases. Higher Varroa mite loads are correlated with the lower potential ability of honey bees to buffer against temperature stress. Our study provides insights into interactions between climate change-related abiotic stressors and their relation to biotic stressors, which underlie a decline in honeybee health.Climate change is associated with warmer and drier weather on average in central California. At the same time, honeybees are being transported from all over the United States to California for the completion of pollination services that ensure increased yields for a variety of crops, resulting in bees experiencing ambient abiotic stressors. Higher temperatures can reduce air quality, further exacerbating honeybee health challenges. We investigated the relationship between higher daily temperatures and poor air quality and demonstrated associations with incidences of pests , which is tied to immune system strength; however, a higher gene expression level of Vitellogenin (Vg) is tied to oxidative stress. There was a positive relationship between Varroa mites and N. ceranae pathogen loads, and a negative correlation between Varroa mites and Heat Shock Protein 70 (HSP70) gene expression, suggesting the limited ability of mite-infested colonies to buffer against extreme temperatures. Histological analyses did not reveal overt signs of interaction between pathology and abiotic stressors, but N. ceranae infections were evident. Our study provides insights into interactions between abiotic stressors, their relation to common biotic stressors, and the expression of genes related to immunity and oxidative stress in bees.Climate change-related extreme weather events have manifested in the western United States as warmer and drier conditions with an increased risk of wildfires. Honeybees, essential for crop pollination in California, are at the center of these extreme weather events. We associated the maximum daily temperature and air quality index values with the performance of colonies placed in wildfire-prone areas and determined the impact of these abiotic stressors on gene expression and histopathology. Our results indicate that poor air quality was associated with higher maximum daily temperatures and a lower gene expression level of Prophenoloxidase ( Apis mellifera) is crucial for pollination in natural and agricultural ecosystems, with annual economic contributions estimated to be over $200 billion globally [Ongoing changes to climatic conditions are resulting in frequent extreme temperature fluctuations and a rise in weather-related natural disasters, which, in turn, affect ecosystems, and the species within . Wildfirglobally ,9 and ovglobally ,11. In rglobally ,14,15,16globally ,17. For globally . Beekeepglobally ,20, decrglobally ,22.Another direct consequence of extreme weather events  in relatVarroa destructor, affect brood and adult bees, and chronic infections from the pathogen, Nosema ceranae, affect adult bees and are critically important. The challenges from these biotic stressors may be compounded as bees adapt to abiotic stressors resulting from changing weather patterns. Our goal in this study is to assess the relationship between two main abiotic factors—temperature and air quality—and explore their relation to the two above-mentioned biotic stressors. Specifically, we investigated how daily maximum temperatures and poor air quality relate to each other and to N. ceranae and V. destructor: the chronic biotic stressors. While N. ceranae is relatively less virulent, it suppresses the immune system and causes energetic stress and metabolic dysregulation that could potentially be compounded by other stressors [Varroa mites, on the other hand, are central to declines in bee health as they nutritionally deplete the honeybee by directly feeding on them [Of the several biotic stressors that affect honeybees, parasitic mites, such as tressors ,16,30,31 on them  and vect on them .Heat Shock Protein 70 (HSP70), a general marker for the ability of bees to buffer against stress from extreme temperature changes [Prophenoloxidase (ProPO), a marker for immune upregulation in response to disease stress [Vitellogenin (Vg), a general physiological stress marker known to be a master regulator with ties to oxidative stress, immune functioning, development, and metabolism [N. ceranae incidence as secondary measures of the potential adverse impacts of temperature and poor air quality. Histologic imaging, which involves the microscopic evaluation of tissues and cells, has more recently gained the attention of honeybee researchers [We then assayed the gene expression of stress biomarkers that are activated in response to different stress pathways with the goal of gaining mechanistic insight into the interactions between abiotic and biotic stressors as they impact honeybee health. We measured the gene expression for  changes , Prophene stress  and Vitetabolism . We furtearchers ,40,41,42https://www.farmersalmanac.com/long-range-weather-forecast, accessed on 15 February 2023) and the air quality index (AQI) for each sampling date was noted from AirNow.gov .This study was conducted from June to November of 2021 with 12 standard Langstroth hives consisting of a total of 18 frames and a 5.68 L  feeder. The hives were equalized in strength and were placed near areas that were known to be prone to wildfires, including six different locations in the Napa, Yolo, and Solano counties of central California . Hives iVarroa mite loads for each colony were also measured via the standard alcohol wash method [Varroa mites in the hive. Colonies with mite levels at over 3% of the threshold received mite control treatments [The monthly, detailed assessments included recording the Frames of Bees (FOB), the presence of a laying queen as indicated by the presence of eggs, and measuring the amount of brood, pollen, nectar, honey, and the amount of empty space on a frame, on a scale of 0–10. h method . Approxieatments . The fraIn-hive and forager bees were placed in separate 50 mL falcon tubes and were thawed, macerated, and homogenized in 5 mL of nuclease-free water  per 30 collected bees, using a 50 mL disposable tissue grinder . The bee homogenate was then aliquoted as a 200 µL volume into three different 1.5 mL microcentrifuge tubes, which were stored in a −80 °C freezer until further analysis.Nosema infection loads was adapted from [Nosema and the identification of the species from a melt curve analysis were performed following [N. apis and N. ceranae positive control as well as a negative control. The samples were run in the Biorad CFX384 qPCR thermocycler  in technical duplicates. If the standard deviation between duplicates was greater than one, the samples were re-run. The amount of Nosema DNA that was quantified was converted to the total number of Nosema copies using the standard curves provided [DNA extraction to quantify ted from . Brieflyollowing . The 25 provided . All primer set sequence information used for gene expression analysis can be found in Heat Shock Protein 70 (HSP70), Vitellogenin (Vg), and Prophenoloxidase (ProPO), were quantified relative to the reference gene Ribosomal Protein S5 (RpS5). The following qPCR protocols were used for each target. For the quantification of the Vg and RpS5 expression, the final volume of the reaction was 10 µL which consisted of 5 µL of qPCR 2x Promega  master mix (no ROX), 0.2 µL (10 µM) of either the Vg-F or Vg-R primers or the RpS5-F and RpS5-R primers, respectively, including 2 µL of template cDNA, and 2.6 µL of nuclease-free water. The qPCR thermocycler program was: 95 °C for 5 min, 45 cycles of 95 °C for 10 s, and 60 °C for 20 s, with a final extension step of 72 °C for 30 s [A blend of random hexamers and an oligo dT SensiFAST cDNA Synthesis Kit  was used following the manufacturer’s instructions to generate cDNA from the extracted RNA standardized to 700 ng. A 20 µL cDNA synthesis reaction was carried out involving 4 µL of a 5x TransAmp Buffer, 1 µL of reverse transcriptase, and 15 µL of extracted RNA. The relative gene expression of a total of three targets, for 30 s . For Profor 30 s . For HSPfor 30 s . Each ruMethods were adapted from Cook et al. (In press). Briefly, honeybees were collected and stored in 10% neutral buffered formalin (NBF) and were fixed for a minimum of 24 h. The head, thorax, and abdomen were separated using spring scissors  or a steel surgical blade . The head and thorax were subsequently sectioned transversely, and the abdomen was sectioned in a sagittal orientation along the midline using a carbon steel surgical blade . Blades were replaced with at least every three samples to prevent dulling, and all tissues were placed with their cut surface down in either one or two tissue cassettes (Tissue-Tek). All cassettes were stored in 10% NBF until further routine laboratory processing, including routine paraffin embedding, sectioning at 4 µM sections, mounting onto glass slides, and stained with hematoxylin and eosin (H&E). Two consecutive sections of all three body segments were mounted onto a single glass slide for light microscopic evaluation and subsequent image analysis. Based on the results of molecular analysis for gene expression, targeted groups were evaluated via light microscopy and image analysis. For microscopic evaluation, all body segments and tissues were assessed, and any lesions were documented. For image analysis, the total surface area for hypopharyngeal glands located dorsal to the central nervous system within the head section was calculated, as was the mandibular gland surface area. For mandibular glands, if both left and right glands were in the plane section of the slide, then both sides were measured and divided by two to obtain an average for one side. If only one side was in the section, then just that half was measured. The surface area of the glands was measured using Adobe Photoshop (v 24.1.1).Nosema. Bees were sampled monthly from June to November 2021. Six sites were sampled from June to September, and 10 nurse and 10 forager bees from each site were processed . One site was discontinued in September as the colonies died, and the remaining five sites were sampled similarly in October and November (100 bees for each time point). Finally, each bee was histologically evaluated for Nosemosis to compare the prevalence diagnosed histologically with the molecular quantification of N. ceranae load, air quality index, maximum daily temperature, the normalized gene expression of HSP70, ProPO, and Vg, FOB, Varroa mite counts, adult bees, the amount of the brood, the amount of honey, the amount of nectar, the amount of pollen, and the amount of empty space in the hive) did not follow a normal distribution. All count data were over-dispersed. Therefore, a generalized linear negative binomial regression model was used where the date of collection, the apiary sampled, and whether the bees collected were foragers or in-hive bees were included in the full model as random effects. The apiary, date, and age class of the bee had significant random effects when analyzing the gene expression of biomarkers for N. ceranae and Varroa mite loads. These random effect variables were then removed from the models due to the resultant lower AIC values when analyzing relationships between diseases and gene expression markers.All statistical analyses were carried out using JMP Pro v. 16  or GraphPad Prism . Data visualization was carried out using GraphPad Prism . All numeric variables  in relationship with gene expression markers and diseases were accounted for in a multivariate Spearman rank correlation analysis.N. ceranae load was analyzed within foragers and in-hive bees in relation to the other numeric variables using a negative binomial generalized linear regression. For N. ceranae and Varroa mites, the date and apiary had significant random effects on the previous negative binomial regression analysis. Therefore, N. ceranae and Varroa mite loads were further analyzed across apiaries and time with Wilcoxon pairwise comparisons after applying relevant post hoc adjustments for multiple comparisons.The Vg, ProPO, and HSP70), the date had a significant random effect in the previous negative binomial regression analysis. Therefore, each gene target was further analyzed across time with Wilcoxon pairwise comparisons.For each gene expression target (N. ceranae and Varroa mites), and gene expression measures . Lastly, a multivariate Spearman rank correlation analysis was performed to determine the association between the detailed assessment measurements , environmental measures , the disease and pest loads . For the quantitative analysis of the histologically determined surface area of hypopharyngeal glands and mandibular glands, Student’s p = 0.027; ProPO and Vg genes   . Among fperature . Air quaperature .N. ceranae and Varroa mites, were related to the gene expression levels of ProPO and HSP70  that were near apiaries that did not receive any hive management measures to control these stressors . Varroa mite loads were positively associated with ProPO gene expression within forager bees . However, there was a negative relationship between Varroa mite loads and forager bee HSP70 gene expression  were all positively correlated with one another and negatively correlated with empty space in the hive. Pollen was positively correlated with adult bees, the frames of bees, and brood. Varroa and N. ceranae loads, levels ProPO, Vg, HSP70). No trends or significant differences in gland sizes were identified for any group comparison  on honeybee health parameters, including common biotic stressors—response ; therefooa mites . In geneoa mites  in honeyVarroa mites were found to be correlated with a potentially lowered ability of foragers to buffer against high temperatures. The lowered expression of HSP70 genes in our analysis suggests exacerbated challenges for honeybees if atmospheric temperatures continue to move toward the extremes in the future. Heat shock proteins were highly conserved across organisms and upregulated in response to temperature and other stressors. These HSP proteins chaperone the assembly, disassembly, folding, and unfolding of protein complexes so that their function can be preserved under stress [HSP70 gene expression, temperature stress, when combined with drought, may reduce pollen production in plants, thus limiting pollen availability for foraging bees [Furthermore, r stress . The roling bees ,53. ThusN. ceranae or Varroa mites is not necessarily associated with a weak colony but instead may be dependent on other factors related to abiotic parameters, implying that colony-level indicators might not always be reliable. These discrepancies could relate to the differences in the seasonality of N. ceranae infections, where spore loads peak in the spring and are lowest in the fall [N. ceranae loads in the spring, but we detected the highest levels in the fall and relatively stable levels throughout the rest of the beekeeping season, suggesting a chronic colony-level infection with this pathogen. The constant higher temperatures and poorer air quality year-round in the Central Valley of California during the study years might suppress the immune system of honeybees, as indicated by the lowering of the ProPO gene expression. This lowered expression could explain the higher disease loads observed year-round in this climate in comparison to more temperate climates, where N. ceranae levels are more seasonal [N. ceranae here is similar to that reported in Spain, where N. ceranae is known to be particularly virulent [N. ceranae infections in Spain are found year-round in colonies with a lack of seasonality for this pathogen [Our results on the colony performance metrics indicate that the presence of the fall , but wheseasonal . The seavirulent . Spain ipathogen . Thus, opathogen . N. ceranae in forager bees versus in-hive bees is also supported by previous findings [N. apis in any of the samples based on the melt curve analysis, supporting the notion that N. ceranae is displacing N. apis throughout the world [ProPO gene expression in forager bees, suggesting that these bees, in an unfavorable environment, may have a decreased ability to combat infections and mount a strong immune response. Poor air quality showed a positive relationship with Vg expression in forager bees and increased Vg through upregulations, which is known to protect workers from oxidative stress, suggesting that poor air quality further stresses honeybee foragers. However, this trend does not hold in nurse bees which may be because they typically already have high levels of Vg expression [ProPO and HSP70 gene expression and not with any of the other stressors. These physiological changes in forager bees from poor air quality exposure may be further exacerbated by maximum daily temperatures, which have been shown to have a strong positive association with poor air quality [The higher amount of findings . This difindings . It is ohe world . Our respression . The nur quality . Our resVarroa is known to suppress the immune system in newly emerged bees [ProPO gene expression in forager bees. Varroa mites and N. ceranae were also positively correlated in this study. Therefore, this and other diseases present in Varroa-infested hives may likely cause a net increase in the innate immune response of forager bees. Supporting this notion, Varroa is known to vector viruses such as the Deformed Wing Virus (DWV) [ProPO expression [ProPO gene in forager bees with a heavy disease burden. Thus, ProPO could serve as a dependable immune marker gene that reflects the ability of bees to fight and fend off potential infections.Despite evidence that ged bees , we founus (DWV) , which bpression . TherefoVarroa mites being negatively correlated with HSP70 gene expression in forager bees, there is a strong likelihood of further hampering the ability of bees to tolerate extreme temperatures [Varroa mites were found to have significantly lower HSP70 gene expression levels [Varroa mite infestation caused a decrease in HSP70 gene expression [Varroa is known to vector virulent viruses, including fungal diseases such as stone brood, feed on fat body and hemolymph, as well as suppress the immune system [Varroa mite loads in hives is indicative of some cautious optimism that the higher temperatures predicted in the future for this region might have benefits, as previous research demonstrated that Varroa mites were indeed sensitive to higher temperatures [Varroa mite loads. However, this comes with several caveats, depending on the levels of high temperatures that honeybees can tolerate. However, it can be noted here that there are several heat shock proteins involved in proteostasis, and our study did not measure the capacity of bees to handle heat stress. Bees are known to have a slightly higher tolerance to high temperatures compared to Varroa mites, but it is also likely that this small beneficial impact could become minimal over an extended time as the parasite adapts to changing environmental conditions [Climate change for our study region is projected to lead to sustained increases in maximum daily temperatures in the future , which ieratures . Furthern levels ; howeverpression . Varroa e system ,33,48,66eratures . Bee heanditions . N. ceranae infections is dependable and the loads detected followed similar trends to the molecular quantification analysis, the relationships between high temperature and poor air quality with expression levels of stress genes were weak. These abiotic stressors did not result in immediate signs of pathological damage to hypopharyngeal or mandibular glands that are indicators of honeybee health [Histological analyses are gradually regaining center stage in research related to honeybee health, but our analyses show that histologic imaging may not be reliable to measure the impact of abiotic stressors on honeybees. While the pathological damage from e health . In summary, poor air quality is a serious environmental stressor impacting honeybees . Air pol


Leishmania genus, poses significant challenges in treatment, including administration difficulty, low efficacy, and parasite resistance. Novel compounds or associations offer alternative therapies, and natural products such as oregano essential oil (OEO), extracted from Origanum vulgare, have been extensively researched due to biological effects, including antibacterial, antifungal, and antiparasitic properties. Silver nanoparticles (AgNp), a nanomaterial with compelling antimicrobial and antiparasitic activity, have been shown to exhibit potent leishmanicidal properties. We evaluated the in vitro effect of OEO and AgNp-Bio association on L. amazonensis and the death mechanisms of the parasite involved. Our results demonstrated a synergistic antileishmanial effect of OEO + AgNp on promastigote forms and L. amazonensis-infected macrophages, which induced morphological and ultrastructural changes in promastigotes. Subsequently, we investigated the mechanisms underlying parasite death and showed an increase in NO, ROS, mitochondrial depolarization, accumulation of lipid-storage bodies, autophagic vacuoles, phosphatidylserine exposure, and damage to the plasma membrane. Moreover, the association resulted in a reduction in the percentage of infected cells and the number of amastigotes per macrophage. In conclusion, our findings establish that OEO + AgNp elicits a late apoptosis-like mechanism to combat promastigote forms and promotes ROS and NO production in infected macrophages to target intracellular amastigote forms.American tegumentary leishmaniasis, a zoonotic disease caused by the  Leishmania amazonensis is one of the main causative agents of American tegumentary leishmaniasis (ATL), which is a zoonotic disease that affects humans and several species of wild and domestic animals and is characterized by chronic inflammation of the skin and mucous membranes [Lutzomyia spp., and its pathogenesis depends on factors such as the virulence of the parasite and the host’s immune response [embranes . ATL is response .® and Pentostam®), which have been used since 1920 [The current treatment of ATL is based on the elimination of amastigote forms, with pentavalent antimonials  has been used in medicine for at least six millennia because of its efficiency in preventing microbial infections. Silver nanoparticles (AgNp) have been shown as potential antimicrobials, causing disruption of the cell integrity, inhibiting ATP synthesis, enhancing ROS production leading to oxidative stress, and disturbing the process of its replication and cell division . RecentlL. amazonensis, both in free promastigote forms and intra-macrophagic amastigote forms.Different therapeutic strategies for the treatment of leishmaniasis patients have been explored, including new natural and synthetic compounds, a combination of compounds, and nanomaterials ,20. TherL. amazonensis (MHOM/BR/1989/166MJO) were maintained in culture medium 199  supplemented with 10% fetal bovine serum (FBS) , 1 M HEPES buffer, 1% human urine, 1% L-glutamine, streptomycin and penicillin , and 10% sodium bicarbonate. The cell culture was maintained in a B.O.D at 25 °C in a 25 cm2 culture flask. In all experiments, promastigote forms in the stationary growth phase were used (5-day culture).Promastigotes of The BALB/c mice were kindly supplied by the Carlos Chagas Institute/Fiocruz-PR, Curitiba, Brazil. The animals were kept under sterile conditions in the Vivarium of the Department of Pathological Sciences of the State University of Londrina until they reached an approximate weight of 25–30 g and age between 6–8 weeks, with controlled light and temperature. The study was approved, and the animals were used according to the rules of the Animal Experimentation Ethics Committee of the State University of Londrina (UEL) nº 8595.2018.89.The OEO was obtained from Ferquima Industry and Commerce of Essential Oils . This oil (batch 224) was extracted by steam distillation, and its density (0.954 g/mL) and composition  were described in a technical report. A 50% OEO stock solution was prepared in dimethylsulfoxide (DMSO) . The maximum concentration of DMSO in the tests was 1%.Fusarium oxysporum was grown in a medium containing malt extract (2%) and yeast extract (0.5%) at 28 °C for 6 consecutive days. The grown biomass was filtered and resuspended in sterile water. Approximately 10 g of the fungal suspension was transferred to a conical flask containing 100 mL of distilled water, maintained for 72 h at 28 °C. AgNO3 was added , and the solution was maintained for several hours at 28 °C for the synthesis of the nanoparticles. The recovered silver nanoparticles were characterized through scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), energy dispersive spectroscopy (EDS) demonstrated by Picoli et al. (2016) [The biogenic silver nanoparticles (AgNp-bio) were obtained according to Durán et al.  ,22. Brie. (2016) , nanopar. (2016) , the dyn. (2016) , plasmon. (2016) .L. amazonensis (106) were treated with different concentrations of OEO , AgNp-bio  and the proportions of the OEO + AgNp associations: 80%/20% , 60%/40% , 50%/50% , 40%/60%  ,and 20%/80% . The parasites were evaluated in a Neubauer chamber after 24 h of treatment. As a negative control, L. amazonensis promastigotes kept in an untreated culture medium were used, and as vehicle control, DMSO 0.1% was used.The promastigote forms of 5 cells/mL) were recovered from the peritoneal cavity with ice-cold PBS supplemented with 3% FBS and then cultured in 24-well plates with 500 µL of RPMI 1640 medium and 10% FBS for 24 h at 37 °C and 5% CO2. The adherent cells were incubated with different concentrations of OEO , AgNp-bio , and the proportions of the association: 80%/20% , 60%/40% , 50%/50% , 40%/60%  and 20%/80%  for 24 h in the same conditions. Subsequently, the cells were washed with PBS, and MTT (5 mg/mL) was added to the wells, followed by further incubation for 4 h. The MTT product  was diluted with 300 µL of DMSO, transferred to a 96-well plate, and read on a spectrophotometer  at 550 nm. As a negative control, untreated macrophages were used, while the positive control was cells treated with 4% H2O2. The results were expressed as a percentage of viability compared to the control group and calculated using the following formula: % (viable macrophages) = sample from the treated group/mean from the untreated control) × 100.To evaluate a possible cytotoxic effect of OEO, AgNp-bio, and the associations of both, the MTT assay -2,5-diphenyltetrazolium bromide) (Sigma-Aldrich) was performed, which measures the metabolic activity of the mitochondria, as described by Mosmann (1983). Macrophages (5 × 1050) and cytotoxicity concentration of 50% of the peritoneal macrophages (CC50) of each proportion of the OEO/AgNp association. For this, the association of OEO/AgNp was evaluated in the following proportions:  that correspond to the doses of . Thus, was constructed an isobologram for anti-leishmania activity and one for the viability of macrophages, where on the x-axis of the graph the AgNp dose was placed and on the y-axis the OEO dose, drawing an additivity line between the values of the isolated substances, wherein points below the line indicate a synergistic effect, points on the line indicate an additive effect, and points above the line indicate an antagonistic effect described by Zhao, Wientjes, and Au (2004) [The interaction dynamics of AgNp and OEO were studied by the method of fixed proportions described by SEIFERT and CROFT, 2006 , to obtau (2004) .50 combined OEO/IC50 OEO alone) +  for activity in promastigotes and CI =  +  for cytotoxicity on peritoneal macrophages, where CI < 1, =1, and >1 indicate a synergistic, additive, and antagonistic effect described by Chou and Talalay (1984) [The value and efficiency of this association can be demonstrated through mathematical calculations using tools such as the combination index (CI) previously described by Hall, Middleton, and Westmacott (1983) . The CI y (1984) .L. amazonensis (106) were treated with the association in the chosen dose (OEO 9.6 μg/mL and AgNp 0.08 μg/mL) and incubated for 24 h. Afterward, the parasites were collected by centrifugation, washed in PBS 0.01 M pH 7.2, and fixed by immersion in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. For evaluation of morphological changes by SEM, promastigotes were fixed with glutaraldehyde adhered to coverslips covered with poly-L-lysine for 60 min. Afterward, they were washed with 0.1 M sodium cacodylate buffer dehydrated in increasing concentrations of ethanol (30–100%), dried at a critical point by replacing ethanol with CO2, coated with gold, and analyzed using a high-resolution double beam electron microscope FEI SCIOS.Promastigote forms of 2 in 0.1 M sodium cacodylate buffer at room temperature and protected from light. After, the samples were washed with 0.1 M sodium cacodylate buffer, dehydrated in increasing concentrations of acetone (50–100%), included in EPON resin, and polymerized at 60 °C for 72 h. Ultrathin sections were made in an ultramicrotome, deposited on a copper grid, and contrasted with uranyl acetate and lead citrate for 20 and 10 min, respectively. The analysis was performed using a JEOL JEM 1400 transmission electron microscope.For evaluation of ultrastructural changes by TEM, promastigotes fixed with glutaraldehyde were transferred to microtubes, washed three times with 0.1 M sodium cacodylate buffer, and post-fixed in a solution of 1% osmium tetroxide, 0.8% potassium ferrocyanide, and 10.0 mM CaCl6) were treated with an association at the chosen dose (OEO 9.6 μg/mL + AgNp 0.08 μg/mL) for 24 h. After, the parasites were washed with PBS and loaded with 10 µM of the probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Sigma-Aldrich) and incubated in the dark for 30 min at 25 °C. As a positive control, 4% H2O2 was used for 30 min. ROS was measured as an increase in fluorescence caused by the conversion of the non-fluorescent dye to the highly fluorescent 2′,7′-dichlorofluorescein (DCF), in a spectrofluorimeter , at excitation and emission wavelengths of 488 and 530 nm, respectively.Promastigote forms (103PO4) 5%) was added. After 10 min of incubation at room temperature, the samples were placed in 96-well microplates. A standard curve was made using serial dilutions of NaNO2, and the absorbance was determined at 550 nm in a microplate reader .Nitrite levels (NO) were determined by the Griess method as an estimation of the nitric oxide produced. Briefly, 60 μL aliquots of supernatants from the anti-promastigote or anti-amastigote assay were recovered, and 60 μL of Griess reagent (1% sulfanilamide and 0.1% of naphthyltylamidine amino acid in orthophosphoric hydrochloride (H6) were treated with an association at the chosen dose (OEO 9.6 μg/mL and AgNp 0.08 μg/mL) for 24 h to assess the potential of the internal mitochondrial membrane (ΔΨm). The treated parasites were washed and incubated with 2.5 μM tetramethylrhodamine ethyl ester (TMRE) (Sigma-Aldrich) for 30 min at 25 °C and analyzed in a spectrofluorimeter  at excitation wavelengths of 480 and emission of 580 nm.Promastigote forms (106) treated with an association at the chosen dose (OEO 9.6 μg/mL + AgNp 0.08 μg/mL) were harvested and washed twice in PBS and stained with 10 μg/mL of Nile red (Sigma-Aldrich) for 30 min at 25 °C. The presence of cytoplasmic lipid bodies was detected in a spectrofluorimeter  at excitation and emission wavelengths of 530 and 635 nm, respectively.Promastigote forms . The parasites were washed twice in PBS, incubated with 5 μL of monodansilcadaverine (MDC) (Sigma-Aldrich) for 1 h at 25 °C, and analyzed using a spectrofluorimeter  at excitation and emission wavelengths of 380 and 525 nm, respectively.6) treated with the combination at the chosen dose (OEO 9.6 μg/mL + AgNp 0.08 μg/mL) for 24 h were washed and resuspended in 100 μL of 1x assay buffer , followed by adding a mixture containing 1 μL of annexin V/FITC  and 1 μL of propidium iodide (PI) (Santa Cruz Biotechnology). Data analysis was performed using a BD Accuri™ C6 Plus flow cytometer. A total of 10,000 events were acquired. Cells negative for annexin V and PI were considered viable, cells stained with annexin V (positive or negative for PI) were considered apoptotic, and cells positive for PI (and negative for annexin V) were classified as necrotic [Promastigote forms (10necrotic .5) were cultured in 24-well plates containing glass coverslips, incubated with 500 μL of RPMI 1640 medium for 24 h at 37 °C and 5% CO2. The adhering macrophages were infected with promastigote forms of L. amazonensis (2.5 × 106) for 2 h. After infection, the non-internalized promastigote forms were removed by washing with PBS, and the adherent cells were treated with the combination of the compounds of the chosen proportion (OEO 9.6 μg/mL and AgNp 0.08 μg/mL), RPMI 1640 medium (negative control), 0.1% DMSO (vehicle), amphotericin B 1 µM (positive control) for 24 h at 37 °C and 5% CO2. Then, the cells were stained with methylene blue eosin solution according to Leishman (Leishman’s dye) . A total of 20 fields were analyzed by immersion at 1000x magnification using an optical microscope .Peritoneal macrophages from BALB/c mice  at excitation and emission wavelengths of 488 and 530 nm, respectively.Macrophages infected with amastigote forms were treated with the dose of the chosen proportion (OEO 9.6 μg/mL + AgNp 0.08 μg/mL) for 24 h. After this period, the cells were washed with PBS and loaded with 10 µM of Hp-value ≤ 0.05 was considered statistically significant.The statistical analyzes were determined by ANOVA, followed by the Tukey test for multiple comparisons. Three independent experiments were carried out, each with datasets in triplicate. The data were expressed as mean ± standard error of the mean. The data were analyzed using GraphPad Prism statistical software . 50) values of the separate compounds, which were the concentration of 16.0 ± 0.05 μg/mL to OEO and 0.2 ± 0.06 μg/mL to AgNp (After 24 h of treatment with different doses of OEO (3.12–100 µg/mL) and AgNp (0.1–10 µg/mL), we verified that the compounds reduced the viability of promastigote forms by 7.61, 36.11, 46.37, 54.03, 86.7, 98.11% for 3.12, 6.25, 12.5, 25, 50, 100 µg/mL of OEO and 33.87, 61.56, 96.95, 100 and 100% for 0.1, 0.5, 1.0, 5.0, 10.0 µg/mL of AgNp, respectively. From these results, it was possible to calculate the half maximal inhibitory concentration , P2 = 7.4/0.06 (±0.04), P3 = 8.0/0.10 (±0.02), P4 = 5.1/0.09 (±0.03), and P5 = 2.5/0.13 (±0.03) OEO/AgNp μg/mL , and the results showed that the proportions P2 = 60%/40%, P4 = 40%/60%, and P5 = 20%/80% of OEO/AgNp presented a CI > 1 , P2 = 90.48/0.75 (±0.02), P3 = 105.90/1.32 (±0.03), P4 = 82.01/1.53 (±0.02), and P5 = 41.06/2.05 (±0.03) μg/mL OEO/AgNp), and the CI value was calculated, with antagonistic results showing CI > 1 , SEM and TEM were performed to determine the morphological and ultrastructural changes induced by the treatment in promastigote forms. Untreated parasites showed normal characteristics compatible with an elongated body, flagellum proportional to body size, smooth and intact cell surface, and well-preserved structures A,E. MeanBased on previous results, we decided to evaluate the production of ROS and NO, the main microbial molecules, in OEO-AgNp-treated parasites. Our results demonstrated that the association induced an increase in both ROS and NO in promastigotes when compared with the control A,B.As TEM analysis indicated mitochondrial damage in treated parasites, we also aimed to confirm that parasite mitochondria were affected by the treatment. As expected, we found a decreased total TMRE fluorescence of 21.7% in the treated group when compared to the control, indicating a loss of mitochondrial membrane potential C.Analysis by TEM also showed an accumulation of lipid-storage bodies in the cytoplasm; thus, we investigated their presence in parasites treated with the association by staining with Nile red, a fluorescent dye with a high affinity to neutral lipids . Our resAdditionally, it was observed by TEM that treatment with association triggered the intense formation of autophagic vacuoles in promastigotes. Thus, we also evaluated whether the treatment induced an increase in autophagic vacuoles, which may precede cell death. Parasites treated and marked with MDC presented an increase in fluorescence of 180% concerning the control, suggesting the formation of autophagy vacuoles E.Knowing that OEO + NpAg treatment induces ROS and NO production, which leads to mitochondrial dysfunction, lipidic bodies storage, and autophagic vacuole formation, we aimed to identify the type of death triggered by this cascade of processes. For this, we performed the co-labeling of annexin V and propidium iodide (PI). Our data showed that the proportion of viable parasites decreases in OEO + NpAg-treated promastigotes, while the annexin V+ and annexin V+/PI+ subpopulation enhanced significantly when compared to the control A,B, indiWe also evaluated the promastigote size, where we found that the OEO + NpAg treatment was able to significantly reduce the size of parasites when compared to the control C,D, confSince amastigotes are intracellular forms, we investigated the effect of the association on infected macrophages. For this, the production of NO and ROS in these cells was evaluated. The results showed a significant increase in NO and ROS compared to the control .p ≤ 0.0001), similar to the positive control, AmB, which induced a reduction of the 46% ± 1.39 (p ≤ 0.0001) when compared to the control. With regard to the number of amastigotes per macrophage, the association has also shown a similar effect to the standard drug (AmB), presenting a reduction of 30.7% (p ≤ 0.01)  .Leishmania spp. parasites, without side effects, is increasing. OEO exhibited a potential leishmanicidal effect on L. amazonensis promastigote forms, resulting in a combination of autophagic, apoptotic, and necrotic events [The current treatment of leishmaniasis has some flaws, such as difficulty in administration, toxicity leading to adverse effects, and parasite resistance, which drives us to search for new treatment strategies for leishmaniasis . In thisc events .The use of nanotechnology has also shown to be a promising strategy, since it is a delivery system that carries drugs to specific targets ,23,33. MIn our study, an important synergistic leishmanicidal effect of the association of OEO + AgNp was demonstrated, since some proportions showed CI values < 1, and in the isobologram, some proportions were below the additivity line. Similar results were observed by Scandorieiro et al. (2016) , which sL. amazonensis. Previous studies using different natural or synthetic compounds on L. amazonensis promastigotes reported similar changes to those observed in our study [SEM and TEM showed that the association induced several morphological and ultrastructural changes in promastigotes of ur study ,9,10,12.Leishmania spp. are its single mitochondria, and the survival of this parasite depends on the perfect functioning of this organelle [We also showed the likely mechanism by which this association exerts its antileishmanial effects. We initially focused our studies on investigating alterations in mitochondria. Notable characteristics of rganelle .We observed an increase in the production of ROS in treated promastigotes. ROS are molecules derived from the incomplete one-electron reduction in molecular oxygen, and high concentrations might induce oxidative damage . The assL. amazonensis mitochondria have been published, showing changes in ΔΨm [Increases in oxidant species induce mitochondrial dysfunction, such as mitochondrial swelling and alterations in the mitochondrial membrane potential ΔΨm . In thiss in ΔΨm ,8,10,41.L. amazonensis were exposed to 4-hydrazinoquinoline analog, these results being compatible with those found in our study.Since mitochondrial dysfunctions, such as mitochondrial depolarization, induce an increase in lipid bodies, the association of OEO + AgNp also increased lipid droplets formation in the cytoplasm, revealed here by the Nile red and TEM . AntinarL. amazonensis, revealed by electron microscopy, and all of the morphological, ultrastructural, and biochemical alterations induced by the association suggest apoptosis-like cell death.The mitochondrial depolarization associated with the accumulation of lipid bodies in the cytoplasm is an event characteristic of the occurrence of apoptosis-like cell death. It is well described in the literature that apoptosis is characterized by several morphological features, among which are cell rounding, cell shrinkage, chromatin condensation, nuclear fragmentation, ultrastructural modifications of cytoplasmic organelles, and plasma membrane modifications with the maintenance of its integrity membrane ,44. Our In this way, we used phosphatidylserine, a phospholipid naturally present in the inner plasma membrane leaflet that switches to the outer leaflet during apoptosis; annexin V, a marker that binds in phospholipid classes ; and PI,The presence of autophagy vacuoles was also observed in our study by MDC labeling. The increase in the formation of autophagic vacuoles can be mainly induced by the increases in cellular ROS levels ,31,38. AL. amazonensis amastigotes forms and reduced the number of infected macrophages [Amastigotes are obligatory intracellular parasites localized in parasitophorous vacuoles of phagocytic cells of the host . AgNp-Birophages .L. amazonensis, since a reduction in the percentage of infected macrophages and the number of intracellular parasites was observed. An increase in ROS and NO in infected macrophages was also observed after treatment with the association. Several studies also describe similar results, demonstrating that the probable mechanism of death of Leishmania spp. is through molecules such as ROS and NO released by infected macrophages [In our study, we observed the anti-amastigote effect of the OEO + AgNp association in macrophages infected by rophages ,9,10.L. amazonensis was not observed by Fanti et al. (2018) [However, the increase in ROS and NO in infected macrophages with amastigotes of . (2018) , which u. (2018) ,35,36 anL. amazonensis through the association of OEO and AgNp, accomplished via metabolic, morphologic, and structural events that induce late apoptosis. In addition, this combination exhibited a remarkable reduction in cytotoxicity in peritoneal macrophages while concurrently eliciting a potent leishmanicidal effect in intracellular amastigotes, possibly via the induction of ROS and NO. These results serve as a powerful impetus to continue our efforts in unraveling the intricate mechanisms of action underpinning the death of this parasite. This combination therapy study determined the mechanisms involved in in vitro OEO + AgNp association against L. amazonensis-infected macrophages and highlights a new and innovative therapeutic approach that can be explored as a candidate for future investigations in drug development in leishmaniasis treatment.Our research findings reveal a strikingly synergistic leishmanicidal effect in vitro on promastigote forms of


We examined the surface wettability, morphology, composition, oil absorption capacity, oil/water separation performance, flux rate, chemical stability, and mechanical stability of the S.P membrane. Our findings indicate that the developed CQD-based S.P membrane possesses excellent S.P properties, displaying high water contact angles of 163° and low water sliding angles of 1°. The membrane demonstrated superior oil absorption capacity, separation efficiency, and flux rate towards three different oils—petroleum ether, n-hexane, and silicone oil. Petroleum ether has the highest separation efficiency (99.5%), and flux rate , while silicone oil has the lowest. However, silicone oil has the highest absorption capacity (218.9 g/g) and petroleum ether has the lowest (194.8 g/g). For the absorption capacity and separation efficiency, a one-way ANOVA test was conducted. The statistical analyses revealed significant differences in absorption capacity and separation efficiency for the three oils, highlighting the efficacy of the superhydrophobic membrane for tailored oil/water separation. Additionally, the S.P membrane exhibited good mechanical (the membrane maintains its superhydrophobicity until an abrasion length of 850 cm) and chemical stability (the membrane maintains its superhydrophobicity in pH range 1–13), withstanding abrasion and immersion in solutions of varying pH values. The CQD-based S.P membrane shows great potential as a promising material for oil/water separation applications, with excellent performance and stability under various environmental conditions.The efficient separation of oil and water is a significant challenge worldwide due to the increasing frequency of industrial oily wastewater. Previous work by our group utilizes biological metal–organic framework-based superhydrophobic (S.P) textile fabric for oil/water separation. However, this system is limited due to the low mechanical stability, so there is a need for producing a more robust S.P membrane for oil/water separation. In this study, we report on the synthesis of carbon quantum dots (CQD) from banana leaves via a hydrothermal process and their application in producing a robust S.P coating on textile fabric for oil/water separation. The CQDs were characterized using various techniques including TEM, XRD, absorbance spectroscopy, and the BET method. The TEM images showed that the CQDs were circular in shape with a size of 4.4 nm, while the XRD micrograph indicated that the CQDs were crystalline in nature. The UV–vis graph showed a peak at a wavelength of 278 nm, suggesting strong absorption in the ultraviolet region. The BET-specific surface area of the prepared CQDs is 845 m Modern human society faces a critical and difficult requirement for a sustainable supply of clean water. The discharge and leakage of oily waste materials into several water streams  have become a significant challenge due to the industrial revolution ,2. The pVarious conventional methods have been utilized to separate mixtures of O-W. These methods include physical techniques such as ultrasonic separation, dissolved air floatation, centrifugation, and gravity separation, as well as chemical techniques such as flocculation separation, neutralization, and coagulation ,8,9,10. Researchers have conducted extensive studies on wettable membranes inspired by nature for O-W separation ,16. ReceS.P materials have demonstrated significant potential in various applications, including corrosion protection of metals and alloys, drag reduction, self-cleaning, anti-icing, and O-W separation ,19,20,21In recent times, different techniques such as plasma etching, chemical vapor deposition, the hydrothermal method, electrodeposition, and atomic layer deposition process have been employed to create S.P membranes ,24,25. HCarbon quantum dots (CQDs) are nanomaterials with carbon skeleton structures that have functional groups on their surfaces and are often spherical in shape and less than 10 nm in size ,28. CQDsFluorinated chemicals, such as fluorocarbon molecules or fluorosilane, are commonly used to create S.P coatings due to their extremely low surface energy . HoweverPrevious work by our group utilized metal–organic framework-coated textiles for oil/water separation . HoweverThe experiment employed analytical-grade sulfuric acid, sodium hydroxide, n-hexane (n-H.E), petroleum ether (P.E), silicone oil (S.O), bromothymol blue, and stearic acid (S.A), which were purchased from Chematek company (Egypt). The pristine textile fabric (TF) and banana leaves were bought from a nearby marketplace.The typical procedure involved washing 2 g of banana leaves with distilled water twice and air-drying them. The dried leaves were then finely powdered using a mortar and pestle. The resulting powder was mixed with 30 mL of distilled water and stirred at room temperature for 15 min. The solution was then transferred into a Teflon autoclave and heated at 200 °C for 3 h. After cooling to room temperature, a carbonaceous solution was obtained and centrifuged at 10,000 rpm for 15 min to remove larger and undissolved particles, and the supernatant solution was collected. The supernatant solution was then passed through a 0.22 μm syringe filter, resulting in a light brown solution that was further diluted with ultrapure water for subsequent use.A circular TF measuring 10.5 mm in diameter was placed in a solution of carbon quantum dots (CQDs) and left to soak for one hour. The TF was then air-dried for 2 h at room temperature and subsequently dried for 2 h at a temperature of 100 °C. Next, the TF was submerged in an ethanolic solution containing 0.01 M S.A for 0.5 h. The resulting superhydrophobic textile fabric, TF@CQD@S.A, was air-dried for 2 h at room temperature and then dried for 2 h at a temperature of 60 °C.The absorption behavior of the prepared CQD was investigated using a UV–vis spectrophotometer with a scan range of 200 to 400 nm. Before measuring the spectra, the samples were diluted in distilled water. All measurements were conducted at room temperature. The size and shape of CQS were determined using a transmission electron microscope . A Bruker D2 Phaser X-ray diffractometer was utilized to perform an X-ray diffraction examination using Cu K radiation with a monochromatic source. The composition of the prepared CQD was analyzed using Fourier-transform infrared spectroscopy (FTIR) (model: Bruker Tensor 37 FTIR). The Brunauer–Emmett–Teller method  was used to investigate the textural properties of the prepared CQD.To examine the morphological changes in the TF following S.P coating, a JEOL JSM-200 IT scanning electron microscope (SEM) was utilized. The elemental composition of the TF before and after S.P coating was analyzed using Fourier-transform infrared spectroscopy (FTIR) (model: Bruker Tensor 37 FTIR). We utilized an energy-dispersive X-ray spectrometer  to determine the elemental composition of the CQDs, and the TF before and after S.P coating. The water contact angle (WCA) and water sliding angle (WSA) were measured with a Rame-hart WCA instrument (model 190-F2) using 5 µL water droplets, and the reported WCA values are the average of four tests conducted at different locations on the prepared STF surface. The pH of the water droplets was adjusted using sulfuric acid and sodium hydroxide.To evaluate the mechanical stability of the prepared STF, an abrasion test was performed. The STF was placed on 800-mesh sandpaper and dragged with a weight that exerted a pressure of 5 kPa, and the WCA and WSA were measured every 5 cm. The chemical stability of the STF was assessed by immersing several fabricated samples in solutions with a pH range of 1 to 13 for one hour, and the impact on the WCA and WSA values was evaluated. The reported mechanical and chemical stability data represent the average of three tests and the standard deviation error bar is represented in the graphs.0 (g). The STF was then placed in oil or organic solvents until it reached adsorption saturation, and it was reweighed again as M1 (g). The adsorption capacity  of the STF was calculated using Equation (1) [When evaluating absorbing materials, absorption capacity is a crucial factor in practice. To determine the adsorption capacity of the STF, a typical absorption test was conducted. Initially, the weight of a clean STF was recorded in the air as Mtion (1) :Q = M1/M0) and after (m1) separation was measured to determine the separation efficiency (η) using Equation (2) [To assess the oil/water separation performance of the STF, a mixture of 20 mL organic solvent and 20 mL water (bromothymol blue was added to the distilled water to provide color) was poured onto a self-built gravity-driven laboratory device. While the TF as a filtration membrane allows the passage of both oil and water, the STF as a filtration membrane allows the passage of oil only and so separation of O-W mixture. Upon introducing oily wastewater into the separation system, the STF facilitated the passage of oil, which accumulated in the lower container, while the water was retained in the upper container. The weight of the oil before (mtion (2) .η =  was employed .Flux = 2) is the contact area, and t (h) represents the recorded oil permeation time. The oil absorption capacity and oil/water separation performance tests were conducted three times and the standard deviation error bar is represented in the graphs. A schematic illustration of the synthesis of CQD and fabrication of STF and its utilization in the separation procedure is presented in Here, V (L) denotes the volume of oil permeation, S  of 0.05 was chosen to assess the results. If the p-value was less than this significance level, it was considered statistically significant, leading to the rejection of the null hypothesis.The statistical analysis for the absorption capacity and separation performance tests for the prepared STF toward the three oils was performed using descriptive statistics and a one-way analysis of variance (ANOVA) test. The data obtained for each trial  of absorption capacity and separation performance for each oil were recorded and analyzed. The control group consisted of pristine textile fabric without any superhydrophobic coating. Descriptive statistics were used to calculate the mean and standard deviation of the absorption capacity and separation performance for each oil. This helped in summarizing the data and understanding the differences in absorption capacity between the superhydrophobic fabric and the control group. To determine if there were significant differences in the absorption capacity and separation performance between the three oils, a one-way ANOVA test was conducted. The null hypothesis (H0) stated that there were no significant differences between the means of the groups, while the alternative hypothesis (HA) hypothesized significant differences between the means. The ANOVA results provided an F-value and a 2 adsorption/desorption isotherm. 2/g, the pore volume is 0.33 cm3/g, and the mean pore diameter of 1.62 nm. When CQDs are grafted onto a substrate, a larger BET surface area of the CQDs will result in the creation of more roughness on the substrate surface (the roughness is the parameter for creating an S.P surface). The yield of prepared CQDs is 13.5%.The CQDs were characterized using TEM, XRD, absorbance spectroscopy, and the BET method, −1 is likely due to the presence of O-H stretching vibrations [−1 and 2861 cm−1 correspond to C-H stretching vibrations, while the peak at 2072 cm−1 is likely due to C≡C stretching vibrations [−1 and 1442 cm−1 correspond to C=C stretching vibrations, while the peak at 1126 nm is due to C-O stretching vibrations. The peaks at 754 cm−1 and 550 cm−1 correspond to C-H bending vibrations [FTIR is a commonly used spectroscopic technique that provides information on the chemical bonds present in a material. The FTIR results for CQDs, TF, and STF are shown in brations . The peabrations . The peabrations .−1 is attributed to the N-H stretching vibrations. The peaks at 2930 cm−1 and 2862 cm−1 are due to the symmetric and asymmetric stretching vibration of CH. The peak at 1643 cm−1 is likely due to C=C stretching vibrations. The peaks at 1305 cm−1 and 1159 cm−1 are due to the C-N stretching vibration. The peak at 989 cm−1 is attributed to the bending of C-H. The peak at 764 cm−1 is due to the out-of-plane bending of N-H while the peak at 595 cm−1 may correspond to C-H out-of-plane bending vibrations [The FTIR results for the TF show various peaks which correspond to various functional groups that are present in the TF. The peak at 3414 cmbrations .The FTIR results for the STF show similar peaks to that of CQDs and TF with a small shift in peak position indicating that the TF is successfully grafted with CQDs and stearic acid.The EDX technique is utilized to investigate the composition of the CQD, TF, and STF, The morphology of both the TF and STF membrane was examined using SEM, as depicted in To assess the wetting behavior of the TF and STF, the WCA and WSA were measured. The TF exhibited hydrophilic properties, with a WCA value equal 0 degrees. This caused water droplets to adhere to the surface without sliding off, even when the fabric was tilted. In contrast, the STF demonstrated remarkable S.P properties, with a WCA value of 163° ± 0.7° and a WSA value of 1° ± 0.1°.To evaluate the stability of the STF, its absorption capacity towards the three oils was measured ten times. After each cycle, the oil absorption capacity was determined, and the STF was compressed to ensure complete desorption of the organic solvent. The absorption capacity of the STF towards the three oils remained constant after ten cycles of absorption and desorption. This suggests that the STF is stable and can be reused multiple times without losing its oil absorption capacity. The prepared STF has superior absorption capacities compared to previously known absorbents ,42.p-value is less than the significance level (α = 0.05), indicating that there is strong evidence to reject the null hypothesis. This means that there are significant differences in absorption capacity between the three oils.The separation efficiency of the STF decreased gradually with the number of cycles, and the separation efficiency reached a minimum value of approximately 95% after ten separation cycles. This decrease in separation efficiency could be due to the accumulation of oil on the surface of the STF, which can clog the pores and reduce its ability to repel water. Additionally, repeated cycles of absorption and desorption can cause wear and tear on the coating, leading to a decline in its performance over time. The O-W separation efficiency of the STF is higher than that of previously reported absorbents ,44.p-value is less than the significance level (α = 0.05), indicating strong evidence to reject the null hypothesis. Thus, it can be concluded that there are significant differences in separation performance between the three oils.The mechanical stability of the developed STF was examined by evaluating the impact of abrasion length on the WCA and WSA on the STF, as shown in The chemical stability of the developed STF under different pH conditions was studied, −2 h−1, followed by n-H.E with a value of 13,100 L m−2 h−1, and S.O with a value of 11,900 L m−2 h−1. This suggests that the membrane has higher permeability towards P.E due to its lower viscosity and surface tension and viscosity compared to the other oils.The flux rate of the developed STF towards three different oils, namely n-H.E, P.E, and S.O, was examined. The results indicate that the flux rate of the STF towards P.E is the highest among the three oils, with a value of 13,500 L m−2 h−1, n-H.E to 12,600 L m−2 h−1, and S.O to 11,100 L m−2 h−1. This decrease in flux rate could be attributed to the accumulation of oil on the surfasce of the coating, which can clog the pores and reduce their permeability. The flux rates of the STF are superior to those of many previously reported absorbents [The impact of repeated separation cycles on the flux rate of the membrane was investigated. The results reveal that after ten separation cycles, the flux values for the three oils have slightly decreased, with the flux rate for P.E decreasing to 12,850 L msorbents .The separation of oil and water is an important challenge due to industrial oily wastewater. Prior work by our group used metal–organic framework (MOF)-coated textiles fabric for oil/water separation. However, the mechanical stability was low. In this work, a robust CQDs-based S.P coating was successfully fabricated on textile fabric for O-W separation. The CQDs were prepared using banana leaves as a raw material by the hydrothermal method.2/g, a pore volume of 0.33 cm3/g, and a mean pore diameter of 1.62 nm. The surface composition, morphology, wettability, O-W separation performance, oil absorption capacity, flux rate, mechanical stability, and chemical stability of the membrane were investigated. The results indicate that the developed STF membrane exhibits excellent S.P properties, with a high WCA of 163 degrees and a low WSA of 1 degree. The membrane also shows good oil absorption capacity, separation efficiency, and flux rate towards three different oils, namely n-H.E, P.E, and S.O. The P.E has the highest separation efficiency and flux rate, while the S.O has the lowest. On the other hand, the S.O has the highest absorption capacity, and the P.E has the lowest. The statistical analyses were performed using a one-way ANOVA test on both the absorption capacity and separation efficiency data. The obtained p-values were significantly lower than the predetermined significance level, indicating robust evidence supporting the presence of significant differences among the three oils. These findings highlight the effectiveness of the superhydrophobic membrane for oil/water separation, with varying performance depending on the type of oil. This information is crucial in the development of efficient and tailored separation materials for specific oil/water separation applications. The membrane also demonstrates good mechanical and chemical stability, with the ability to withstand abrasion and immersion in solutions of different pH values for varying immersion times.The prepared CQDs were characterized using multiple techniques, including transmission electron microscopy (TEM), X-ray diffraction (XRD), absorbance spectroscopy, and the BET method. TEM analysis revealed that the CQDs exhibited a circular shape with an average size of 4.4 nm. XRD analysis indicated that the CQDs possessed a crystalline structure. The UV–vis spectrum exhibited a pronounced peak at 278 nm, suggesting strong absorption in the ultraviolet region. Furthermore, the BET analysis determined that the prepared CQDs had a specific surface area of 845 m