Foliar Zn application significantly increased the Zn concentration and bioavailability in wheat grain and flour. If rural households consumed Zn-biofortified flour instead of self-cultivated flour or flour purchased from supermarkets, 257–769 or 280–838, 0.46–1.36 million or 0.50–1.49 million disability-adjusted life years (DALYs) lost, respectively, could be saved in Quzhou County and China. Amounts of 2.3–12.0 million and 5.5–22.6 billion RMB could be obtained via Zn-biofortified flour in Quzhou County and China, respectively. The current study indicates that Zn-biofortified flour via foliar Zn application is a win-win strategy to maintain the yield and combat human Zn deficiency in rural households in China. More health and economic benefits could be obtained in rural household dependent on wheat flour purchased from supermarkets than in those dependent on self-cultivated wheat flour.Zinc (Zn) malnutrition is a common health problem, especially in developing countries. The human health and economic benefits of the replacement of conventional flour with Zn-biofortified wheat flour in rural household diets were assessed. One hundred forty-five wheat flour samples were collected from rural households in Quzhou County. Then, field experiments were conducted on wheat at two Zn levels (0 and 0.4% ZnSO As an essential micronutrient, zinc (Zn) plays a vital role in crop production and human nutrition. At present, Zn deficiency, also called hidden hunger, is a common public issue worldwide, contributing to many health problems . Zn defiThe widespread occurrence of Zn malnutrition in humans mainly arises from a low dietary intake of Zn . Current−1 in China, indicating a wide gap to the target value of 40 mg kg−1 , consisting of 10 townships and 342 administrative villages. However, a previous survey has revealed that 39% of the children in Handan city suffers from Zn malnutrition  to analyze the current Zn intake based on samples of the wheat flour consumed daily in rural households in Quzhou County, (2) to study the effects of foliar Zn application on the Zn concentration and bioavailability in wheat grain and flour under diverse agricultural practices in Quzhou County, and (3) to comprehensively assess the health and economic impacts of the replacement of conventional wheat flour with Zn-biofortified flour in rural household diets in Quzhou County and China via scenario simulation.Field experiments were conducted at 16 locations in Quzhou County  in the North China Plain . Quzhou 4 · 7H2O, w/v). Foliar Zn was applied twice as follows: the first spraying was conducted at the early milk stage, and the second spraying occurred a week later. A Tween  solution was applied at 800 L ha−1 in the foliar Zn application treatment. Spraying was conducted on cloudy days or after sunset under windless conditions across all 16 locations. The area of each plot was 100 m2 at all 16 locations. In addition to foliar Zn application, routine cropping practices were implemented in the present study, including various seeding rates, fertilizer application, herbicide sprays, and irrigation.Each location included two treatments: the control treatment  and foliar Zn application , and the rate of flour extraction was ~75%, which is similar to the general flour extraction rate in China market . (2) Flour purchased from the supermarket by the rural households (n = 21). These two wheat flour types were separately consumed by the rural households in Quzhou County as part of their daily diets.A total of 145 wheat flour samples was also randomly collected from 21 villages (including the 16 test locations) to estimate the current Zn intake via wheat flour consumption in the rural households in Quzhou County. We divided the sources of wheat flour into two groups: (1) flour milled from wheat grain that was self-cultivated by the rural households . The micronutrient concentrations in the digested solutions were determined via inductively coupled plasma optical emission spectroscopy . Standard wheat grain material (IPE182) was acquired from the Wageningen Evaluation Programs for Analytical Laboratories  and used to ensure consistency and quality. The PA concentrations in the wheat grain and flour samples were analyzed according to a previous study (−1 diethylene triamine pentaacetic acid (DTPA)  . The soid (DTPA) .A trivariate mathematical model of Zn absorption was adopted to predict the Zn bioavailability :−1), TDZ and TDP are the total daily dietary Zn (mmol Zn day−1) and PA (mmol PA day−1), respectively, AMAX is the maximum Zn absorption (0.091), KR is the equilibrium dissociation constant of the Zn-receptor binding reaction (0.680), and KP is the equilibrium dissociation constant of the Zn-PA binding reaction (0.033) (−1) as the sole source of Zn and phytate for adults  , while tr adults , which iex ante assessment tool to estimate the burden of micronutrient malnutrition and the health impact of micronutrient-biofortified wheat flour  is an at flour . The curat flour . Infantsat flour . The totat flour . The staectively . Based oeach day . The daieach day .The following equation was adopted to calculate the economic benefit of Zn-biofortified wheat flour:Economic benefit = total DALYs saved × PCNI – Zn fertilizer cost.−1) according to Wang et al. . Similarly, the average of the above three parameters (the Zn and PA concentrations and the estimated Zn bioavailability) for the two sources of wheat flour collected from rural households were also compared via independent t-tests (P < 0.05).Excel 2010  and SPSS software (version 26.0) were used for the calculations and statistical analysis. The effects of foliar Zn application on the Zn and PA concentrations and the estimated Zn bioavailability in wheat grain and flour were assessed via one-way analysis of variance (ANOVA) followed by independent −1 under the control and foliar Zn treatments, respectively.Foliar Zn application imposed no significant effects on the wheat grain yield at any location . The aveThere was a large variation in the Zn and PA concentrations and estimated Zn bioavailability in the wheat flour samples irrespective of the source. The Zn concentration in the self-cultivated wheat flour was significantly higher than that in the wheat flour purchased from supermarkets. However, no significant differences occurred in the PA concentration and estimated Zn bioavailability between the two sources of wheat flour .−1, respectively (−1) was obtained at 12 field locations due to foliar Zn application. Foliar Zn application imposed no significant effects on the PA concentration in wheat grain and flour, and the PA concentration in wheat grain was much higher than that in flour across the 16 locations  (−1) (−1 (n = 188), which was lower than our result of 7.1 mg kg−1. Different manufacturers and production and processing methods may explain this difference. In addition, the current study demonstrated that the Zn concentration in the flour purchased from supermarkets was lower than that in the flour self-cultivated by the rural households. The reason for this may be that the extraction rate of the flour purchased from supermarkets is lower than that of the flour self-cultivated by rural households, and more Zn is lost during milling   and high−1) (−1) , which l−1) (−1) , 34. Wei−1) (−1)  reported milling .Considering Zn homeostasis in human intestines, the Zn bioavailability in wheat flour is more important than the Zn concentration in wheat flour. The observed difference in wheat flour Zn bioavailability between the self-cultivated flour and the flour purchased by rural households suggests a relatively high vulnerability to Zn malnutrition of rural households dependent on wheat flour purchased from supermarkets . Rosado −1 due to foliar Zn application, which matches the biofortification target of Zn in wheat grain.In the present study, foliar Zn application imposed no significant effect on the wheat grain yield, which is consistent with previous studies , 38. In As a store of P and energy, PA plays an important role in plant growth and development and functions as an antioxidant and anticarcinogen in the human body . UnfortuThe current study suggested that the estimated Zn bioavailability in wheat grain and flour was significantly enhanced by foliar Zn application, which is consistent with the findings of Li et al. . Based oConsidering the role of wheat flour as a staple food in Quzhou County and the estimated Zn bioavailability in Zn-biofortified flour being significantly higher than that in the self-cultivated flour or the flour purchased by rural households, substitution of conventional wheat flour with Zn-biofortified flour in rural household diets could highly alleviate Zn malnutrition.−1) (−1)  in Quzhou County was greater if Zn-biofortified flour replaced the flour purchased from supermarkets than that if the flour produced on rural household farmlands was replaced. This occurs because the Zn concentration and bioavailability in the flour produced on rural household farmlands were higher than those in the flour purchased from supermarkets. Our results also revealed that the reduction in the burden of Zn deficiency in China (12.33–13.43% under the pessimistic scenario and 36.87–40.18% under the optimistic scenario) due to foliar Zn application was larger than that due to biofortification in India (2–12%) and Pakistan (5–33%) , 50. In −1)  and Chin−1) (−1) , a relat−1) (−1) . In summOur farm field experiment demonstrated that foliar Zn application effectively increased the Zn concentration and bioavailability in wheat grain and flour irrespective of the agricultural management practices in Quzhou County. Zn-biofortified wheat flour provided ~57% of the daily Zn requirement. Based on the defined scenarios, more health and economic benefits could be obtained by the replacement of self-cultivated flour or flour purchased from supermarkets with Zn-biofortified wheat flour in Quzhou County and China. Therefore, foliar Zn application is a win-win agronomic strategy to maintain the yield and combat human Zn deficiency in rural households in China.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.B-GY: resources, data curation, and writing—original draft. Y-ML, X-XC, W-QC, and T-BD: investigation. C-QZ: conceptualization, writing—review, and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Over the past few years, rechargeable aqueous Zn-ion batteries have garnered significant interest as potential alternatives for lithium-ion batteries because of their low cost, high theoretical capacity, low redox potential, and environmentally friendliness. However, several constraints associated with Zn metal anodes, such as the growth of Zn dendrites, occurrence of side reactions, and hydrogen evolution during repeated stripping/plating processes result in poor cycling life and low Coulombic efficiency, which severely impede further advancements in this technology. Despite recent efforts and impressive breakthroughs, the origin of these fundamental obstacles remains unclear and no successful strategy that can address these issues has been developed yet to realize the practical applications of rechargeable aqueous Zn-ion batteries. In this review, we have discussed various issues associated with the use of Zn metal anodes in mildly acidic aqueous electrolytes. Various strategies, including the shielding of the Zn surface, regulating the Zn deposition behavior, creating a uniform electric field, and controlling the surface energy of Zn metal anodes to repress the growth of Zn dendrites and the occurrence of side reactions, proposed to overcome the limitations of Zn metal anodes have also been discussed. Finally, the future perspectives of Zn anodes and possible design strategies for developing highly stable Zn anodes in mildly acidic aqueous environments have been discussed. Renewable energy supplies have drawn worldwide attention because of the scarcity of fossil fuels and the increasing global warming ,2,3. Nev−1) than most organic solvents 2@Zn anodlectrode c. In add3 coating to control the Zn deposition  . On the other hand, the battery with the pristine Zn anode had a lower initial capacity of 188 mAh g−1, which remained only 124 mAh g−1 after 1000 cycles. Later, Zeng et al. proposed a similar strategy using conductive CNT scaffolds . T. T3 coatctively) b. The ba 1 A g−1 c. Its cacaffolds . The hig2+ ion flux. As a pioneer in this field, Zhao et al. proposed a solid-state interphase comprising of polyamide (PA) and zinc trifluoromethane sulfonate (Zn(TfO)2) . . 123]. Uer 200 h b. This sWith regard to PVDF, Liu et al. synthesized a MOF-PVDF composite coating layer consisting of PVDF and hydrophilic microporous metal-organic framework (MOF) particles . The met3 (BTO) can effectively restrain the Zn dendrite growth (2O)62+) were easily attracted by the O atom in BTO via hydrogen bonding, which facilitated the diffusion of Zn ions and restricted the Zn dendrite formation [−2, 1 mAh cm−2  cormation . In contmAh cm−2 c and formAh cm−2 d. After 2+ was preferentially absorbed by CuO and dispersed evenly on the Zn surface, which was advantageous for Zn nucleation. Furthermore, as illustrated in 2+ distribution on the surface, small Zn nanosheets were consistently developed on the CuO nanowires, as shown in the SEM image  was produced using magnetron sputtering as a scaffold combining a 3D architecture and an interfacial control to efficiently promote the uniform nucleation of Zn metal and reduce the growth of Zn dendrites [2+ transfer and deposition could be accelerated by the desolvation process of Zn2+ with surface chemistry control. The 3D hierarchical conductive carbon scaffold uniformly distributed the electric fields and lowered the local current density. The N-containing functional groups in NC served as nucleation sites to distribute the Zn nuclei uniformly across the electrode surface. As Zn was constantly deposited, an electric charge was generated in the uneven nucleation sites. The carbon scaffold doped with N atoms encouraged the homogenous nucleation and growth of Zn, thus constraining the formation of Zn dendrites. The N atoms generated by the addition of pyrrole lowered the migration energy barrier and promoted the redistribution of Zn ions. Furthermore, the zincophilicity of the NC scaffold was enhanced by the strong electrostatic interaction between the negatively charged pyrrole N sites in the carbon lattice and the Zn atoms, resulting in the homogeneous nucleation and growth of Zn. The negative state of the pyrrole N site can be explained by the formation of σ-bonds between the doped N atoms and the adjacent carbons from which electrons moved toward pyrrole N through the inductive effect. As a result, the assembled half-cell showed a high zinc stripping/plating CE of 98.8%. The NC-Zn symmetrical cell showed stable operation at a high current density of 5 mA cm−2 with an overpotential as low as 11 mV after 210 cycles.Zhang et al. investigated CuO nanowires grown on a Cu mesh (CM@CuO) by anodic oxidation, followed by thermal annealing in air . BecauseEM image b. Zn remmage  for AZIBs in mildly acidic electrolytes are summarized in 2+, and controlling the electric field. Corrosion and HER can be resolved by reducing the chemical activity of H2O and introducing the protective layer on the Zn metal anode. As can be seen from According to the strategies discussed above for modifying the Zn metal surface, the drawbacks must be addressed concurrently to achieve a practical improvement of Zn metal anodes. As for the dendrites, they can be effectively suppressed by increasing the Zn nucleation sites, redistributing the ionic flux of Zn2 evolution. The presence of uneven nucleation sites on Zn metal facilitates uncontrollable charge transfer, which accelerates the generation of an uneven electric field, causing the growth of dendrites. The corrosion of Zn metal anodes can be categorized as self-corrosion or corrosion by an electrochemical reaction. The latter refers to the irreversible Zn consumption due to the removal of Zn from the electrode as well as the side reactions between the electrode and electrolyte. H2 evolution is a complicated process that is affected by the reduction potential, overpotential, surface area, electrolyte pH, and operating temperature. To address these issues, researchers have proposed various modification strategies, which can largely be categorized as (i) shielding the Zn metal from side reactions, (ii) regulating the Zn deposition behavior (controlling the nucleation sites and redistributing the Zn2+ ion flux), and (iii) establishing a uniform electric field.In summary, the major challenges associated with the application of Zn metal anodes in mild aqueous electrolytes and the modification strategies employed to overcome these challenges are discussed to provide an insight into the development of high-performance AZIBs. AZIBs are regarded as promising alternatives to LIBs  owing to their safety, cost-effectiveness, environmental benignity, and high energy density. The main challenges associated with the use of Zn metal anodes in neutral or mildly acidic media are dendrite growth, corrosion, and HDespite the considerable progress made in this field, there is still a long way to go to address the complex challenges discussed thus far. An efficient approach to produce a dendrite-free and highly reversible Zn anode is to strategically tailor the 3D structure of the electrode. The construction of 3D nanostructured Zn metal anodes has not been fully explored yet. Fabricating Zn anodes with hierarchical 3D architectures provides new opportunities for increasing the rate capability and service life of AZIBs because it allows a more uniform distribution of the electric current and provides a large number of reaction sites. The fabrication of 3D Zn architecture can be realized by the coating of Zn metal  on a well-defined conductive 3D framework  or by the selective etching of the sacrificial components in Zn composites. The caveat here is that while the increase in the surface area of the electrode can improve the energy density and suppress the dendrite development, it can accelerate the corrosion of Zn metal. This can be avoided by coating 3D Zn metal with corrosion inhibitors or by introducing organic/inorganic additives in the plating electrolyte. In addition, although the behavior of Zn electrodes in mild aqueous electrolytes differs from that in alkaline electrolytes, the knowledge of the operation of Zn batteries in alkaline aqueous media can provide time-saving guidance for the study of AZIBs. Furthermore, more progress can be made by investigating the previously reported strategies for modifying metal anodes .4 and Zn(CF3SO3)2 are mainly used within a certain concentration range. In most cases, ZnSO4 is preferable because of its relatively low price as compared to that of Zn(CF3SO3)2. However, ZnSO4 suffers from the formation of hydroxide sulfate by-products (Zn4(OH)6SO4·nH2O). Exploring other Zn salts for application under mild aqueous conditions and some appropriate ZnSO4-based additives is another potential approach. Mn-based materials are the most widely used cathode materials. As revealed by many studies on AZIBs cathodes, Mn-based oxides can be operated at high working voltages, but it is still difficult to fully understand their reaction mechanism because of the diverse phase transitions occurring during the reactions. Apart from Mn-based cathode materials, V-based materials and Prussian blue analogs can be potential candidates for application in conjunction with Zn metal anodes for AZIBs. However, in this case, the limitations of these materials  should be considered.In addition to the Zn metal anode, the other components (cathode and electrolyte) of the cell should also be taken into consideration to improve the performance of the anode. The type of electrolyte, electrolyte concentration, and additives are important factors that strongly influence the electrochemical reactions on the Zn metal anode. In particular, in mild aqueous electrolytes, ZnSOFinally, most studies on Zn metal anodes have mainly focused on coin cells or pouch cells. To meet the market demands , these studies need to be expanded to larger cells . In practical applications, unknown challenges that have not been encountered in coin cells can be encountered. The know-how of various cell configurations can narrow the gap between the lab-scale results and the commercialization of AZIBs. Finally, the development of flexible and wearable AZIBs can be an intriguing research topic because of their intrinsic safety, low cost, and facile fabrication (without the need for a glovebox). To realize this, not only should a sufficiently thin Zn metal electrode  be developed, but appropriate components  compatible with Zn anodes should also be carefully designed.


In this study, we evaluated the leaf antioxidative responses of three wheat varieties  treated with two different forms of zinc (Zn), Zn-sulfate and Zn-EDTA, in concentrations commonly used in agronomic biofortification. Zn concentration was significantly higher in the flag leaves of all three wheat varieties treated with Zn-EDTA compared to control and leaves treated with Zn-sulfate. Both forms of Zn increased malondialdehyde level and total phenolics content in varieties Srpanjka and Divana. Total glutathione content was not affected after the Zn treatment. Zn-sulfate increased the activities of glutathione reductase (GR) and guaiacol peroxidase (GPOD) in both Srpanjka and Divana, while glutathione S-transferase (GST) was only induced in var. Srpanjka. Chelate form of Zn increased the activities of GST and GPOD in both Simonida and Divana. Catalase activity was shown to be less sensitive to Zn treatment and was only induced in var. Srpanjka treated with Zn-EDTA where GPOD activity was not induced. Concentrations of Zn used for agronomic biofortification can induce oxidative stress in wheat leaves. The antioxidative status of wheat leaves could be a good indicator of Zn tolerance, whereas wheat genotype and chemical form of Zn are the most critical factors influencing Zn toxicity. Zinc (Zn) is an essential metal for plants, and adequate availability of this micronutrient is vital at all stages of plant development. This micronutrient acts as an enzyme cofactor and thus plays an important role in regulating metabolic processes like the synthesis of nucleic acids and proteins, pollen formation, carbohydrate metabolism, and auxin synthesis ,2. HowevZn fertilizers are widely used in agronomic biofortification to elevate Zn concentrations in grains of wheat and different cereals ,15. AddiThis work aimed to investigate the effects of two different forms of foliar-applied Zn on the leaf antioxidative status of three wheat varieties, var. Divana, a Croatian standard of high quality, var. Srpanjka, a standard for high yield and the most common variety in Croatia, and var. Simonida, a standard for high yield and the most common variety in agricultural production in Serbia. We hypothesized that the antioxidative response would be variety specific, and would be dependent on the applied Zn-form. The results given in this paper provide a better understanding of the possible toxicity of Zn used in agronomic biofortification and antioxidative leaf status as a potential biomarker of its toxicity. Additionally, this study emphasizes the importance of genetic factors of different wheat varieties that determines the variety-specific antioxidative response to Zn treatment.Foliar application of Zn-sulfate did not cause changes in the Zn content in the flag leaves, while the treatment with Zn-EDTA significantly increased the concentrations of Zn in the flag leaves of all three wheat varieties compared to the control plants . VarietyThe lipid peroxidation products, such as MDA, are usually measured as an indicator of oxidative stress. A significant increase in lipid peroxidation levels was showed in flag leaves of varieties Srpanjka and Divana treated with both forms of Zn . A slighIn leaves of all three varieties tested, treatment with Zn in both forms had no impact on the total glutathione (tGSH) content a. The acThe antioxidant enzymes (guaiacol peroxidase (GPOD) and catalase (CAT)) activities were affected due to Zn treatment, although the effects depended on the wheat variety and the Zn form . The actTreatments with both forms of Zn caused an increase in the total soluble phenolics content but only in leaves of var. Srpanjka and Divana. In the leaves of var. Simonida, Zn treatment did not cause significant changes in the content of total phenolics .Agronomic biofortification is a promising strategy for increasing Zn concentration in grains of different cereals . AlthougLablab purpureus), and okra (Hibiscus esculentus cv. Hassawi) treated with Zn [Corchorus capsilaris L. where the addition of EDTA diminished the copper toxic effects and, at the same time, improved copper accumulation Zn [Phytotoxic concentrations of Zn stimulate lipoxygenase activity leading to the induction of lipid peroxidation, one of the most reliable indicators of oxidative stress . In this with Zn ,31. Alth with Zn ,33. Althation Zn . Diaz etation Zn  found siThe results of studies conducted in different transgenic and wild-type plants, Zn accumulators, hyperaccumulators, and Zn-resistant plant genotypes, showed that higher levels of GSH and increased activity of enzymes involved in the metabolism of GSH result in intrinsic resistance to excess HMs ,37,38. GIn this research, Zn treatment did not cause significant changes in the GSH content compared to the control leaves  a. On theCajanus cajan), rice (Oryza sativa), golden bean (Vigna radiata), and corn (Zea mays) treated with different HMs [In our study, foliar application of Zn-sulfate increased GR activity in the flag leaves of var. Srpanjka, while in var. Divana, both sources of Zn resulted in a significant increase in GR activity with a more pronounced effect of EDTA form b. On therent HMs ,46,47,48rent HMs . The autrent HMs . The incrent HMs , as confrent HMs .P. vulgaris) exposed to elevated concentrations of Zn [In our experiment, there was no correlation between the concentration of tGSH and GR activity, which was also found in hydroponic-grown seedlings of beans (ns of Zn .Vigna radiata) [Hordeum vulgare) can be induced by a variety of metals, including Zn. As a stable and efficient response, GST activity is induced under conditions of increased oxidative stress when the basal antioxidant enzymes are exhaust [In addition to the ascorbate-glutathione cycle, high status of GSH is essential for the detoxification effect of GST. It was found that exposure to cadmium causes an increase GST activity in leaves of golden beans (radiata) , while Hradiata)  showed i exhaust .Our results showed that increased activity of GST in the leaves might be the result of treatment with Zn c. In the2O2 [2O2; thus, it is activated only when high levels of ROS are produced [2O2. An increase of total phenolics content was observed in different plant species exposed to HMs [GPOD and CAT are the most important antioxidative enzymes in plants that detoxify H2O2 . Their aproduced . In addiproduced ,54,55. Id to HMs ,56. Ma ed to HMs  found thd to HMs , while td to HMs . Such and to HMs ,60. In tThis research provides a better understanding of the wheat antioxidative defense mechanisms in response to Zn, and could contribute to the Zn-biofortification strategies and the sustainability of wheat production. In conclusion, we showed that concentrations of Zn used in agronomic biofortification can induce oxidative stress in wheat leaves. Leaf antioxidative response depends on the genotype and the chemical form of Zn used in the experiment. This research suggests that the antioxidative status of wheat leaves could be a reliable indicator of Zn tolerance, with var. Simonida being the most tolerant genotype. To understand the regulation of the processes of absorption, remobilization, compartmentalization, and transport of Zn to the grains, gene expression analysis of Zn-transporters should be performed.Triticum aestivum L.) varieties  were grown under field conditions in seasons 2012/13 on Mollic Gleysol in the Danube region . Sowing depth was 4–5 cm with seeding rate 220 kg/ha (Divana) or 260 kg/ha (Srpanjka and Simonida) seeds for 550 spikes/ha (Divana) or 700 spikes/ha . The Mollic Gleysol was calcareous (4.95% CaCO3), moderately alkaline (pHH2O = 8.13), with moderate SOM (3.00%) total Zn content (53.5 mg kg−1) extracted by aqua regia , and plant available Zn content (1.11 mg kg−1) extracted by EDTA [Three winter wheat ( by EDTA .2). The Zn foliar application treatments were as follows:Control without Zn application;−1 Zn in the form of ZnSO4 × 7 H2O (6.6 kg ha−1);Zn-sulfate: application of 1.5 kg ha−1 of Zn in the form of Zn-EDTA (10 kg ha−1).Zn-EDTA: application of 1.5 kg haAll treatments were distributed in complete randomized blocks with three replications of basic experimental plots (size 20 m−1 of solutions (0.25% Zn w/v) or water for control treatment, plus 0.1% (v/v) surfactant, early in the morning between heading (Feekes 10.3) and beginning of flowering (Feekes 10.51). The leaves for analyses were collected by sampling across the whole plot for the average sample.All treatments were applied using 600 L ha3 and H2O2 mixture  in a microwave oven (CEM Mars 6) at 180 °C for 60 min. The concentrations of Zn were determined by ICP-OES (PerkinElmer Optima 2100 DV) using an internal pooled plasma control and the reference material  prepared in the same way as were the plant samples. All samples were analyzed in duplicate, and the results were expressed in mg kg−1 of dry weight.The dried leaf samples were ground into a fine powder using a heavy metal-free ultra-centrifugal mill (Retsch ZM 200). All samples were digested with 10 mL of HNOw/v) solution of trichloroacetic acid (TCA). After centrifugation of the homogenates, the resulting supernatant was mixed with the thiobarbituric acid  prepared in a 20% (w/v) TCA solution. The result of this reaction was the formation of red coloration, whose intensity was determined spectrophotometrically by measuring the absorbance at 532 nm and 600 nm. The absorbance at 600 nm was subtracted from the absorbance at 532 nm due to the correction for non-specific reactions. The amount of MDA was calculated based on the extinction coefficient (ε = 155 mM−1 cm−1) and expressed in nmol per g fresh weight (nmol MDA g−1 FW).The products of lipid peroxidation in wheat leaves treated with Zn were determined with the method of Verma and Dubey  based onw/v) in 5% 5-sulfosalicylic acid solution and centrifuged at 10,000× g for 10 min at 4 °C. The resulting supernatant was transferred to the reaction mixture that contained 100 mM phosphate buffer with 1 mM EDTA (pH 7.0), 0.031 mg mL−1 DTNB, and 0.115 units mL−1 of GR in a final volume of 1.05 mL. The reaction was initiated by adding NADPH at a final concentration of 48 μM. The total amount of GSH was determined by a standard curve of GSH, and the results were expressed as nmol g−1 of FW.Content of tGSH in wheat leaves treated with Zn was determined using a kinetic method based on a continuous reduction of 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) to 5-thio-2-nitrobenzoic acid (TNB) by catalytic amounts of GSH, where GR and NADPH recycle the GSSG . The forg and +4 °C. The resulting supernatants were stored at −80 °C and used for the spectrophotometric determination of the activity of the enzymes CAT (EC 1.11.1.6), GPOD (EC 1.11.1.7), GR (EC 1.8.1.7), and GST (EC 2.5.1.13).Frozen leaf powder (about 200 mg) macerated with liquid nitrogen with the addition polyvinylpyrrolidone (PVP) was extracted for 15 min on ice with the addition of 1 mL of cold 100 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA. Obtained homogenates were centrifuged for 15 min at 20,000× 2O2 was catabolized [2O2. CAT activity was expressed in enzyme units (U) representing the amount of enzyme that catalyzes the oxidation of 1 µmol H2O2 per min at 25 °C and pH 7.0 per g of fresh weight.The total CAT activity was measured by following the decrease in absorption at 240 nm for 2 min as Habolized . The enz2O2 in 0.2 M phosphate buffer (pH = 5.8) [The total GPOD activity was determined spectrophotometrically by measuring the absorbance increase at 470 nm. The reaction mixture contained 5 mM guaiacol and 5 mM HH = 5.8) . The enzThe activity of GR was determined according to the method described by Halliwell and Foyer . The reaThe GST activity was conducted spectrophotometrically by monitoring the formation of the reaction product of conjugation between GSH and 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm ,67. The 2O, 100 µL of Folin-Ciocalteu reagent, and 300 µL of sodium carbonate saturated solution was incubated in a water bath at 37 °C for 1 h. The absorbance of the prepared samples was determined at 765 nm, and total phenol content in ethanol extract was calculated from the calibration curve with the gallic acid used as a standard. Soluble phenolic content in fresh tissue was expressed as mg of GA equivalents (GAE) per g of fresh weight.The ethanol extracts of wheat flag leaves were obtained by extracting 100 mg of frozen leaf powder with 1 mL of 80% ethanol in a bath at 80 °C for 30 min. The total phenol content was determined spectrophotometrically by the method of Folin-Ciocalteu . The rea


This study enriches the fundamental comprehension of Ohmic contact interfaces on the Zn deposition, which may shed light on the development of other metal battery anodes.Zn metal holds grand promise as the anodes of aqueous batteries for grid‐scale energy storage. However, the rampant zinc dendrite growth and severe surface side reactions significantly impede the commercial implementation. Herein, a universal Zn‐metal oxide Ohmic contact interface model is demonstrated for effectively improving Zn plating/stripping reversibility. The high work function difference between Zn and metal oxides enables the building of an interfacial anti‐blocking layer for dendrite‐free Zn deposition. Moreover, the metal oxide layer can function as a physical barrier to suppress the pernicious side reactions. Consequently, the proof‐of‐concept CeO 2+ diffusion kinetics and a reduced Zn nucleation barrier, thus achieving a dendrite‐free and side‐reaction‐free Zn deposition chemistry for significant improvement of the electrochemical performance in both the symmetric cells and full cells.A universal Zn‐metal oxide Ohmic contact interface model is demonstrated to effectively enable improved Zn The dendrite formation may pierce the separator and eventually renders battery failure or even safety hazards. In addition, the continuous accumulation of irreversible by‐products 6·xH2O and H2) not only reduces the Coulombic efficiency (CE) of the cell, but also increases the inner pressure that may inflate the cell to failure. Therefore, solving the dendrite and side reaction issues are of great significance for the commercialization of Zn anodes.Rechargeable aqueous Zn‐ion batteries have garnered an extensive research interest due to their attractive merits of low price, non‐pollution, and rich abundance in the earth's crust.1 Howe electrolyte modulation, and a three‐dimensional (3D) design, have been developed to deal with the above‐mentioned critical problems. In particular, surface modification attracts tremendous attention due to its remarkable protective effect and easy manipulation. The recent endeavors are primarily concentrated on metals, carbon matrix, polymers, metal‐organic frameworks, and metal oxide coatings. Among these candidates, metal oxides show a great potential to effectively suppress the dendrite growth on the Zn anode, which will propel the commercialization of aqueous Zn‐ion batteries. For instance, ZrO2 featuring a high dielectric constant and good chemical stability was demonstrated as an excellent artificial coating layer that provides more nucleation sites and enhanced Zn2+ transport kinetics through “space charge polarization”, which guarantees the uniform Zn stripping/plating and long cycling stability. An ultrathin Al2O3 layer was coated on a Zn anode to enable enhanced corrosion resistance and suppressed dendrite. TiO2 coating was also revealed to be capable of suppressing the corrosion and HER of Zn anode. More recently, Wang et al. revealed that TiO2 with exposed (001) facet exhibits relatively low Zn affinity, thereafter preventing the vertical growth of Zn dendrites and stabilizing the Zn‐electrolyte interface. Despite these great endeavors, however, the underlying mechanism on why and how these metal oxides affect the Zn deposition still remains unclear.To date, a variety of design strategies, such as surface modification,4 elec than many metal oxide semiconducting materials, such as TiO2 (4.4–5.0 eV), WO3 (4.3–4.8 eV), MoO3 (6.2–6.7 eV), and CeO2 (4.3–4.7 eV). The high work function difference could drive electrons flowing from metallic Zn to the oxide semiconductors, thus building an Ohmic contact interface  electrode exhibits a long lifespan up to 1300 h with high CE, and moreover, a full cell based on CeO2@Zn//MoS2 also delivers a brilliant rate capability and long cycling stability.It is important to notice that metallic Zn owns lower work function (3.6–3.8 eV)16 tha Figures . Inspire2 forming a typical Ohmic contact interface from a physics viewpoint  distinctly exhibit linear features, which reveals the non‐rectifying Ohmic contact characteristics. It should be noted that the negative charge accumulation will lead to the establishment of an electron enrichment region at the interfaced metal oxides, which is called an “anti‐blocking layer” herein. When immersed into the electrolyte, the negative charges in the anti‐blocking layer will have crucial effects in attracting the cations but repelling the anions through electrostatic interaction, thus regulating the cation flux for homogeneous Zn deposition . Scanning electron microscopy (SEM) image reveals the nanoparticle morphology of CeO2  technique. As shown in Figure 2 (PDF: 03‐065‐5923), respectively.To demonstrate the practicality of this unique Zn‐metal oxide interface model, as a proof‐of‐concept, mesoporous CeO2 Figure 2a. The lectively.242@Zn anodes. Notably, the pristine Zn exhibits a metallic luster character, while the surface of CeO2@Zn is slight yellow stemming from the intrinsic color of nano‐CeO2. Contact angle measurements were performed to compare the hydrophilia of the two samples. Impressively, the CeO2@Zn electrode delivers dramatically improved electrolyte wettability (63°) compared to the pristine Zn (87°), . The synergistic combinations of good wettability, sufficient sub‐nano transportation channels, and high potential gradient are of significant benefit for improving the Zn2+ diffusion capability.Figure , Figure . The enhng layer.25 In 2+ transference number (tZn2+) was then applied to quantitatively estimate the Zn2+ diffusion capability of CeO2 layer. Here, the Bruce–Vincent method was executed to evaluate the tZn2+ value in the system ; I0 and R0 are the initial current and interface resistance, respectively; Is and Rs represent the steady‐state current and interface resistance, respectively. As displayed in Figure tZn2+ value in the bare Zn system is as low as 0.33, which can be ascribed to the unrestricted migration of SO42− anions compared to hydrated Zn2+. In sharp contrast, the value for CeO2@Zn symmetric cell dramatically increases to 0.77 due to the improved Zn2+ diffusion kinetics and restrained anions transfer, which is highly desirable for high performance battery operation.Where 2 coating layer on suppressing the side reactions, both CeO2@Zn and bare Zn electrodes were soaked in 2 m ZnSO4 for 10 days. As shown in Figure 4SO4(OH)6·xH2O by‐product 6·xH2O species are fundamentally absent  measurements. Remarkably, the CeO2@Zn electrode shows a higher HER overpotential of 0.90 V at 5 mA cm−2 and a higher Tafel slope of 336 mV dec−1, compared to that of the bare Zn (0.78 V and 199 mV dec−1) . The high and stable CE and lower polarization potential electrochemically elucidates the importance of the CeO2 layer on promoting the Zn platting/stripping reversibility and kinetics.The electrochemical plating/stripping behavior of Zn was investigated using asymmetric Zn—Cu and CeO2@Zn and bare Zn electrodes. Figure −2. Impressively, the CeO2@Zn cell delivers a long life‐time up to 1300 h, which is far better than that of the bare Zn (<100 h). In addition, the CeO2@Zn anode shows a small voltage hysteresis of 42 mV with ultrastable voltage–time curves even after 1000 h (inset). In strong contrast, a high voltage hysteresis of 67 mV and a quick short circuit are observed for bare Zn. The similar phenomenon is further identified at high current densities of 1.0  than that of bare Zn (47 mV) at 0.5 mA cm−2, suggesting that the Zn2+ nucleation barrier is significantly reduced. Even at high current densities of 1 and 5 mA cm−2, a similar trend can also be concluded  compared to the bare Zn cells, indicating rapid Zn2+ transference stemming from the mesoporous CeO2 layer. In order to clarify the effect of polyvinylidene fluoride (PVDF) binder on the Zn anode, we also evaluated the electrochemical behaviors of PVDF‐coated Zn anode. As shown in Figure 2+ deposition kinetics. Therefore, the enhanced Zn2+ deposition kinetics of CeO2@Zn originates from CeO2 layer instead of PVDF.Symmetric cells were assembled to evaluate the cycling stability of CeO2 Figure . The com2+ on the anode surface. As shown in Figure 2+. This nucleation mode will cause the subsequent Zn nucleus to aggregate and grow into Zn dendrites, which is typical of the noted “tip effect”. In sharp contrast, the CeO2@Zn electrode manifests a typical 3D diffusion and nucleation process as the planar nucleation in a CeO2@Zn symmetric cell accounts for only 5 s, suggesting the regulated Zn2+ diffusion and nucleation upon deposition.Chronoamperometry (CA) measurement was carried out to fundamentally analyze the deposition behavior of Znigure 2+.9 This2+ nucleation and growth on bare Zn and CeO2@Zn. Figure −2 at current densities of 0.5, 1, and 5 mA cm−2, respectively. After Zn deposition, large protrusions are unevenly distributed on the surface of Zn at 0.5 and 1 mA cm−2, which may be due to the rampant 2D diffusion at low current densities that leads to the accumulation of Zn at the initial nucleus, corresponding well with the electrochemical analysis in Figure −2. These large protrusions can reach a size of 100 µm, which can easily pierce the separator and cause the battery short circuit . More importantly, the specific capacity of CeO2@Zn//MoS2 battery stabilizes at 167 mAh g−1 when the current density goes back to 0.1 A g−1, while the bare Zn//MoS2 cell undergoes dramatic capacity fading. This excellent rate capability can be attributed to the improved Zn2+ diffusion kinetics in the CeO2@Zn/MoS2 battery, as further elucidated by the decreased Rct value . When assembled with the MoS2 cathodes, the CeO2@Zn enables the full cells to manifest a superior rate (96 mAh g−1 at 4 A g−1) and cycling performance. It is believed that our fundamental findings herein will provide a valuable guideline for developing ultrastable metal anodes beyond Zn‐ion batteries.In summary, a universal Zn‐metal oxide Ohmic contact interface model has been established to regulate the homogeneous Zn deposition behavior. The as‐demonstrated CeOThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Contrary to other head and neck subsites, oropharyngeal squamous cell carcinoma (OPSCC) has shown a considerable increase in incidence over the past 20 years. This growing incidence is largely due to the increasing place of human papillomavirus (HPV)-related tumors. HPV-positive and HPV-negative OPSCC are two distinct entities with considerable differences in terms of treatment response and prognosis. However, there are no specific recommendations yet in the therapeutic management of OPSCC patients according to their tumor HPV-status. The aim of this review is therefore to discuss the therapeutic management of patients with OPSCC and the impact of HPV status on treatment selection.Since there is no published randomized study comparing surgical and non-surgical therapeutic strategies in patients with oropharyngeal squamous cell carcinoma (OPSCC), the therapeutic management of these patients remains highly controversial. While human papillomavirus (HPV)-positive and HPV-negative OPSCC are now recognized as two distinct diseases with different epidemiological, biological, and clinical characteristics, the impact of HPV status on the management of OPSCC patients is still unclear. In this review, we analyze the current therapeutic options in patients with OPSCC, highlighting the most recent advances in surgical and non-surgical therapies, and we discuss the impact of HPV status on the therapeutic strategy. Head and neck squamous cell carcinoma (HNSCC) accounts for more than 600,000 new cases each year worldwide and represents the 6th cause of cancer deaths ,2,3. In There has been considerable debate over the last three decades regarding the initial therapeutic management of OPSCC ,11. To dIn the light of the results of larynx preservation programs, the 1990s saw an increased use of non-surgical treatments, for OPSCC, combining radiotherapy (RT) with chemotherapy (CT), commonly referred to as an organ-sparing therapy protocol ,12. ThisThe aim of this review article is therefore to discuss current therapeutic strategies in patients with OPSCC and the potential impact of tumor HPV status.HPV-positive OPSCC is a unique entity both clinically and demographically ,7. HPV-pAt the molecular level, HPV-induced carcinogenesis leads to functional abrogation of p53 and pRb pathways, mediated by the expression of the viral oncoproteins E6 and E7 . E6 bindIn a comprehensive genomic characterization of HNSCC, the Cancer Genome Atlas Network showed that HPV-associated tumors are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1 . By contRegarding clinical presentation, HPV-positive OPSCC are characterized by a frequent discordance between small primary tumor size and significant lymph node involvement . This exMultiple studies demonstrated that HPV tumor status was the main prognostic factor in OPSCC patients ,7,29. ThSecondly, HPV-positive OPSCC are characterized by an improved chemo- and radiosensitivity compared to HPV-negative OPSCC ,30. CompThirdly, HPV-positive OPSCC patients display a lower risk of second primary neoplasia than HPV negative OPSCC patients ,20. The All these differences and particularly the considerable discrepancy in terms of prognosis have led to the creation of two distinct TNM classifications, in the 8th UICC/AJCC staging system, according to the p16 tumor status of OPSCC patients ,37. The Interestingly, several studies demonstrated that, besides p16 tumor status, tobacco consumption was an important prognostic factor in OPSCC patients ,40. IndeAmerican and European guidelines on the management of OPSCC patients do not differ according to the HPV status of the tumor ,43,44. I2 laser and especially robotic surgery  [OPSCC oncologic surgery has undergone an intense transformation over the past 30 years. Although complete tumor removal with free surgical margins remains the cornerstone of surgical treatment, surgical techniques have evolved from radical non-conservative open surgery to functional organ preservation surgery ,46,47. S System) ,50. Tran System) . Some au System) .Similar to primary tumor resection, neck surgical treatment has evolved into more conservative procedures, from radical neck dissection to modified and selective neck dissection . With thAlong with the development of more conservative surgical procedures, considerable advances have been made in head and neck reconstructive surgery. This progress is mainly linked to the development and refinements of microvascular surgical techniques ,53,54. TEarly-stage OPSCC  can be managed by either surgery or RT alone ,44. WhenIn patients with locally advanced  OPSCC treated by upfront surgery, postoperative RT is almost always indicated. A low proportion of patients with a pT3N0 tumor or with a small unique metastatic lymph node in the upper neck without any other adverse pathological feature may avoid postoperative RT ,44. PostNon-surgical treatment of patients with OPSCC is mainly based on definitive RT. RT alone on the primary tumor and neck is sufficient for early-stage OPSCC, whereas it should be potentiated by a systemic therapy for locally advanced OPSCC ,44.Similar to head and neck surgery, RT techniques have largely evolved over the past 20 years. The use of intensity-modulated RT (IMRT) has become the gold standard in patients with OPSCC and has demonstrated an improved preservation of salivary function compared with conventional RT, without compromising locoregional control or survival . IMRT alThree-weekly (100 mg/m²) cisplatin-based concurrent CRT is the gold standard non-surgical treatment of locally advanced OPSCC ,63. In pIn contrast to de-escalation studies conducted in HPV-positive OPSCC, other studies have examined the opportunity to enhance treatment intensity by combining additional therapy with the conventional cisplatin-based CRT ,68. ThesEarly-stage  OPSCC can be treated by either surgery or definitive RT alone. There is no demonstrated survival advantage with primary surgery compared to definitive RT for early-stage HPV-positive OPSCC ,73. IndeWhatever the therapeutic strategy, the overall prognosis of these patients is excellent and the main objective should be therefore to preserve functions and QoL. Consequently, primary surgery should not be the first therapeutic option if the surgeon is not confident that it will provide optimal functional results. This depends mainly on the anatomical subsite and extension of the tumor. If surgery is selected, a transoral approach should be preferred. Indeed, minimally invasive surgery (TORS and elective neck dissection or SNB) has demonstrated promising results in terms of swallowing function and could be an interesting option to avoid late side-effects of RT ,75. SeveAt the opposite, several retrospective studies showed that, for HPV-negative OPSCC, upfront surgery was associated with improved oncologic outcomes, including for early-stage tumors ,15. IndeIn locally advanced  resectable OPSCC, primary surgery is associated with significant functional impairments depending on the anatomical subsite and tumor extensions ,76. Althp = 0.10) [p = 0.002). However, the high rate of successful surgical salvage for locoregional recurrence in the non-surgical group  explained the absence of significant impact on OS [In HPV-positive locally advanced OPSCC, there is no clear benefit in terms of survival of primary surgery followed by adjuvant (C)RT compared with definitive CRT ,72,78. Ip = 0.001 for the whole cohort; HR = 0.74, 95% CI: 0.60–0.91, p = 0.005 in the HPV-negative group; and HR = 0.85, 95% CI: 0.70–1.03, p = 0.098 for the HPV-positive group) [p = 0.01), DSS (p = 0.02), and RFS (p = 0.02), compared with non-surgical treatment  [Conversely, although there is no randomized controlled study comparing surgery followed by adjuvant (C)RT and definitive CRT in patients with HPV-negative locally advanced OPSCC, most recent cohort studies suggested that primary surgery provided a clear benefit in terms of oncologic outcomes in this population ,15,80. D. 42.2%) . Altoget. 42.2%) .HPV-positive OPSCC occurring in smokers (>10 pack-years) exhibit an intermediate prognosis between HPV-positive tumors in non-smokers, which are associated with the best prognosis, and HPV-negative tumors, which are associated with the worst prognosis ,83,84. IIn addition to oropharyngeal tumors invading the pterygoid process, the skull base, the nasopharynx, or the carotid artery, those with a large tongue base involvement crossing the midline have to be classified in this category because primary surgery would lead to unacceptable functional impairment  ,85.Definitive cisplatin-based concurrent CRT is the gold standard treatment for locally advanced unresectable OPSCC, whatever the HPV-status ,86.Patients with HPV-negative unresectable OPSCC bear a poor prognosis with reported 5-year OS rates inferior to 35% ,86,87. AAt the opposite, even with a T4 unresectable tumor, HPV-positive OPSCC patients display satisfactory survival rates, particularly if they are non-smokers ,89. HoweTreatment of recurrent and/or metastatic OPSCC is similar to that of other recurrent and/or metastatic HNSCC. There is no specific recommendation for oropharyngeal tumors and no particularities according to the HPV status ,44,90. HBriefly, salvage surgery, when feasible, is the gold standard therapy for loco-regional recurrence ,92. WherLocal therapy  is a valid therapeutic option for patients with a single metastatic or oligometastatic disease . In otheThe three main perspectives in the treatment of OPSCC can be summarized as follows: 1—to determine the optimal therapeutic strategy between primary surgery and RT alone in patients with early-stage OPSCC; 2—to improve oncologic outcomes in patients with HPV-negative locally advanced OPSCC with an intensified therapeutic regimen that does not raise acute and chronic treatment-related toxicities; 3—to reduce long-term functional impairment and improve QoL in HPV-positive locally advanced OPSCC patients through de-escalation therapeutic strategies without compromising survival.As mentioned previously, primary surgery and definitive RT lead to satisfactory and comparable oncologic outcomes in patients with early-stage OPSCC ,72,73,96Approximately half of patients with HPV-negative locally advanced OPSCC will present tumor recurrence and will die from their cancer . There iGiven the favorable prognosis of HPV-positive OPSCC patients, several studies have investigated the possibility to reduce treatment intensity without compromising oncologic outcomes  ,99,100. The role of tumor HPV-status on therapeutic decision-making in OPSCC patients is not yet well defined. However, the tumor HPV status will have, in the near future, a major impact on the therapeutic management of OPSCC patients . There is no published randomized phase III clinical trial comparing surgical vs. non-surgical therapeutic strategies in OPSCC patients. Nevertheless, there are convergent data supporting the use of primary surgery in patients with HPV-negative OPSCC since it is associated with improved oncologic outcomes, if an acceptable functional result can be reasonably expected. In patients with HPV-negative unresectable OPSCC, cisplatin-based CRT remains the gold standard treatment since recent studies aiming at intensifying therapeutic strategy have failed to improve both oncologic and functional outcomes. In patients with early-stage HPV-positive OPSCC, surgery and RT lead to comparable survival outcomes and treatment selection should be mainly based on treatment-related morbidity and preservation of swallowing function and QoL. In patients with HPV-positive locally advanced OPSCC, upfront surgery plus adjuvant (C)RT is associated with increased morbidity and functional impairment and no substantial gain in terms of survival compared with definitive CRT. In these patients, cisplatin-based concurrent CRT remains the cornerstone of the treatment but research is being undertaken to assess new therapeutic regimens  in order to minimize treatment-related toxicities.


Background: Perceptions of cannabis-related risk are changing, and many are viewing cannabis as harmless despite the biopsychosocial risks. Perceptions of risk have an impact on behavior as individuals who are less likely to view cannabis as risky are more likely to use it problematically. Purpose: This study examined how mental health professionals who use cannabis perceive the risks related to use. Methods: Interpretative phenomenological analysis was utilized to understand how participants made sense of the harm related to personal and client use. Interviews were conducted with a sample of social workers, nurses, and psychotherapists who work with cannabis-consuming clients. Results: Participants reported cannabis use is related to anxiety, relational challenges, impaired driving, psychosis, cognitive impairment, educational/employment dysfunction, and addiction in some users. Conclusion: Assessing risk perceptions among cannabis users can reveal subtle psychosocial problems the user may be experiencing. Mental health workers may benefit from further education regarding cannabis-related physical health harm. Perceptions of cannabis-related risk have been changing across Canada since cannabis legalization in 2018. Many view this drug to be relatively harmless despite the known psychological, physical, and social risks . PerceptAcute cannabis consumption can cause anxiety, panic, cognitive impairment, and psychotic symptoms in some users, as well as motor vehicle accidents among impaired drivers . FrequenPrevious studies have applied rational choice theory to understand cannabis use among adult Canadians and related risk perceptions . This leCannabis use has long been considered a means to relieve stress , and somOnly 2% of lifetime cannabis consumers in Canada have sought professional support for related problems . While mThis study followed a qualitative design informed by interpretative phenomenological analysis (IPA). This approach involves exploring perspectives, meanings, and lived experiences of a specific phenomenon, with emphasis on understanding how participants make sense of their personal and social worlds . Interprn = 7) of Canadian mental health professionals. Convenience and purposive sampling were utilized. Eligible participants (a) were regulated mental health professionals, (b) had direct service experience with cannabis-using clients, and (c) had recreationally consumed cannabis at least once in the past month. The sample consisted of three registered social workers, two nurses (one nurse practitioner), and two registered psychotherapists. The four female and three male professionals reported employment in community mental health, hospital, and private practice settings. They described serving clients including children, youth, families, and adults with a broad range of psychosocial challenges. The study’s methods were approved by a Canadian University Research Ethics Board (REB), and participants provided written informed consent to participate in the study. Guidelines proposed by the REB limited questioning participants around negative personal experiences with cannabis to prevent undue distress. Additional consideration was given to privacy and confidentiality due to the sensitive nature of the subject matter. Participants were de-identified in all possible documentation and given an opportunity to review the findings to ensure quotes could not reveal their identities  disclosed cannabis personally increased their anxiety at times, with Participant 6 stating: “I don't really smoke in public nowadays ‘cause it kind of gives me social anxiety.” Anxiety is clinically related to avoidance, and the potential for cannabis to be used to “avoid painful emotions” and “not allow [clients] to really examine what they’re feeling” was also discussed by Participants 1 and 7.A lot of parents have rules in their home of not using, or kids aren’t allowed in if they’re clearly high … it makes the other parts of just living more challenging. So if they’re not allowed home on Sunday night because they’re clearly high, then it’s harder to have somewhere to sleep at night, and a meal that evening, and get … to school the next day. [Participant 3]Six participants described how cannabis could negatively affect interpersonal relationships. Four reported it could impair one’s “ability to function [and] socially cope” by “limit[ing] your ability to interact with others, ‘cause you can get panicky” [Participant 2]. Participant 7 explained: “I had young clients… getting high with their friends because that’s what everyone’s doing. But they’re having a horrendous, horrific experience while everyone else is enjoying it. But they don’t want to say ‘no’ because of peer pressure.” It was noted by Participant 2 that some clients are reluctant to accept recommendations to reduce or discontinue use because they fear “they’re going to lose connection[s]” with peers. Three informants expressed concerns about the potential for cannabis to “cause some tension in a relationship… if a parent or a spouse really feels strongly that it is not a good thing to be using” [Participant 2]. According to one professional who works with youth:A component of relational challenges identified by participants 4 and 5 was “social stigmatization.” Negative assumptions about the effects of cannabis can result in judgments toward users being “criminal[s]” or “lazy” [Participant 4]. These social perceptions were reported to contribute to self-stigma: “Some people say, like, they’re just getting known as ‘the stoner.’ So, they do have a label of ‘this is who I am.’ … I do wonder [how] that impacts them and how they view themselves” [Participant 2].1 Six participants described “driving while high” as risky, with Participant 6 stating: “Being stoned and driving is obviously a huge no-no.” This behavior was also described in relation to “chronic” use and employment problems as some workplaces require operation of a vehicle and the harm associated with impaired driving can increase when it occurs while at work.Another common risk identified by the professionals was the potential for cannabis-impaired driving.Five participants reported cannabis use “could lead to psychosis” or exacerbate symptoms of schizophrenia. According to Participant 5, “There are certain people that … I’ve recommended probably should stop [using cannabis] because … they were experiencing symptoms that …fell along the schizophrenia [spectrum].” Participant 4 stated: “I’ve had clients where doctors identified marijuana as being the reason for [developing] schizophrenia.” The potential for cannabis to make young people more vulnerable to psychosis or long-term users more resistant to antipsychotic treatment was also discussed.Five participants described how cannabis could affect various dimensions of cognitive functioning. Four expressed concerns about the potential for disrupted adolescent brain development: According to Participant 2: “If they’re under the age of 25, there’s some pretty good literature on brain development in teenagers. So right away, if you’re under 25, I’m going to be giving some education on what happens [to] the brain [while using cannabis].” Three informants described how it could negatively affect attention and concentration, with Participants 5 and 6 personally disclosing: “it decreases my concentration” and “if I smoke too much, my mind starts to wander.” Participants 4 and 7 discussed how cannabis could contribute to “decreased motivation” and reduced “ability to function on a day-to-day basis.” Concerns about impaired memory were noted by Participants 2 and 1, particularly in relation to “retaining information” and “remember[ing] key facts when they’re starting a new role” at work or school. One professional [Participant 6] shared the story of a client with an acquired brain injury who struggled with “managing his confusion … just remembering things was an issue. But when he stopped smoking [cannabis], you know, those issues kind of, like, went away.”The potentially deleterious effects on academic and employment functioning were noted by five participants. Concerns were expressed regarding cannabis impacting clients’ ability to secure, prepare for, and maintain employment. According to Participant 1: “I have had clients that are using daily that I’ve encouraged to reduce their use, especially surrounding when they’re going to be starting a new job.” Others also shared concerns about clients and members of the public “smoking at work,” with Participant 6 explaining, “if they’re chronic [users] then it can affect their ability to work … operate a vehicle [and] interact with others… you forget things, obviously, when you’re at work and stoned.” Several professionals described how cannabis could affect academic functioning, with Participant 3 stating: “Some [students] are skipping school to go and smoke… [then] they have a harder time focusing or, like, being an active participant in school.”I’m really pissed off at myself that I couldn’t [cope] myself and make myself feel better… I’m not using cannabis to get blasted and completely detach. I’m using it … to calm [myself] when I can’t use the available skills in that moment.The potential for cannabis consumption to become a “chronic” habit or addiction was noted by four participants. According to Participant 6: “the risk for addiction is very real… it can be really, really hard to stop.” Participant 7 expressed concerns about clients developing “negative, unhealthy pattern[s]” of consumption or needing to “wake and bake” (smoke after waking in the morning) as signs of dependence. Two participants were personally concerned about developing a “psychological addiction.” Participant 6 shared: “There are times when I tell myself, oh, I shouldn’t do it, but I do it anyway, you know? Um, so it was prominently habitual… sometimes I regret smoking or smoking so much.” Participant 2 shared that sometimes after using it to manage physical or psychological discomfort:The risks identified by the participants in this study are interconnected and reflect how the harms associated with cannabis use impact one another. The potential for this drug to cause or worsen anxiety and psychotic symptoms in some users was noted by most participants, with these effects reportedly contributing to relational difficulties. On occasions when social anxiety or panic is felt after taking cannabis, the consumer may find it difficult to communicate effectively or navigate complex social situations. Difficulties managing stressful interactions can intensify interpersonal anxiety, at times prompting increased cannabis use in an attempt to reduce or avoid that feeling. A cycle of cannabis causing anxiety and anxiety leading to more cannabis use may ensue, with the drug being consumed to manage a condition it may be aggravating. As noted by one participant, the experience of cannabis-induced panic in a social situation where others appear to be enjoying themselves can be isolating, although peer pressure creates barriers to limiting consumption. For those who belong to social circles where cannabis is common, recommendations from professionals to reduce or stop may be met with ambivalence, reluctance, or resistance. This reality produces barriers to treatment for individuals whose anxiety escalates to psychotic symptoms such as paranoia or perceptual disturbances. Cannabis-induced psychosis, panic, and/or “bad trips” can result in hospitalization while wreaking havoc on one’s social and family life.Several participants linked the themes of cognitive impairment and addiction to problems with school, employment, and driving. When cannabis intake becomes a habit or dependence, changes to memory, motivation, attention, and concentration can become entrenched . These dA personal risk/reward analysis was described by almost all the professionals in this study. While cannabis was reported to increase anxiety and hinder cognition in some users and two participants noted concerns about dependence, the majority indicated that the benefits of consumption eclipsed the costs. This group weighed the harms they knew could be incurred against the benefits they perceived and determined that consumption was a reasonable personal choice. Particularly for those managing pain or insomnia, cannabis was reported to be the best option for relief. In studies by Two participants in this study indicated that they sometimes regretted using this drug. One described ambivalence, at times espousing the benefits and at other times wishing they had alternative skills to cope. Another alluded that the negative effects of social anxiety, sleep disturbance, and wandering thoughts overshadowed the benefits despite ongoing use. While The professionals in this study demonstrated an intricate understanding of the psychological, social, familial, and functional risks associated with cannabis use. Their cumulative, off-hand knowledge reflected many of the concerns outlined in a World Health Organization report on nonmedical cannabis use . Their pInterestingly, only one participant mentioned the potential for smoking cannabis to cause physiological damage. While many Canadians acknowledge the risk for pulmonary injury and medical professionals link long-term use to cardiovascular disease, none of the informants noted these harms . This abThis study shed light on how Canadian mental health professionals who use cannabis can reduce stigma and barriers to mental health care for cannabis users. The participants’ responses challenge public perceptions that cannabis use is harmless and emphasize the value of providing fact-based education for clients and families tailored to address their specific concerns. The findings also challenge the notion that cannabis use is inherently irrational and underscore the importance assessing the individualized psychosocial effects of the drug. Through dispelling myths about cannabis and creating an inclusive therapeutic environment, clients can feel comfortable enough to share their problems, motivations, goals, and appraisals of risk. As these professionals shift clinical and public discourse toward the facts and away from unfounded assumptions, barriers to care for cannabis users who are seeking support  can be diminished.This study’s findings suggest assessing perceptions of cannabis-related risk among consumers in clinical settings may help reveal latent psychosocial problems. Examining how cannabis affects anxiety, relationships, driving, psychotic symptoms, cognition, and educational and employment functioning can provide a deeper understanding of lived experiences of harm. Helping clients weigh the perceived risks and benefits can set the foundation for therapeutic approaches that address ambivalence and promote recovery. The findings suggest mental health workers may benefit from education regarding the physical health harms associated with this drug. As mental health professionals and services evolve to adapt to the changing health climate, a need remains for improved access to evidence-informed, destigmatized care for people who use cannabis.This study was limited by time constraints, small sample size, and convenience sampling. Future research using larger samples of professionals and mixed methods approaches may offer more comprehensive understandings of the associations between cannabis use and specific health or clinical outcomes. An analysis of perspectives on stigma, legalization, and barriers to accessing services would have enhanced this study. The findings of this study may not be generalizable to professionals in regions where cannabis is not legal as concerns related to criminal sanctions are likely to be perceived as a risk in jurisdictions where cannabis possession remains a criminal activity. Interviews were conducted prior to widespread awareness of the coronavirus pandemic in Canada and it is unclear how this global crisis may have impacted participant views. This study did not examine choice of cannabis products due to ethical concerns as only legal market products provide validated product information and inquiry into potentially illicit activities was intentionally avoided. There are significant variations in psychoactive effects among various cannabinoids, plant strains, and formulations. Future studies should investigate the perceptions of risk related to specific cannabis products.


TP53, APC, KRAS, and PIK3CA. In total, 16 somatic mutations were detected in the CRC group and in either the IBD or CP group. In addition, our data showed that 51% of total somatic variants were CRC-specific variants. The average number of CRC-specific variants per sample is 2.4. The top genes carrying CRC-specific mutations are APC, TP53, PIK3CA, FBXW7, ATM, and SMAD4. It seems obvious that TP53 and APC genes were the most affected genes with somatic mutations in all groups. Of interest, 85% and 28% of the APC and TP53 deleterious somatic mutations were located in Exon 14 and Exon 3, respectively. Besides, 37% and 28% of the total somatic mutations identified in APC and TP53 were CRC-specific variants, respectively. Moreover, we identified that, in 29 somatic mutations in 21 genes, their association with CRC patients was unprecedented. Ten detected variants were likely to be novel: six in PIK3CA and four variants in FBXW7. The detected P53, Wnt/βcatenin, Angiogenesis, EGFR, TGF-β and Interleukin signaling pathways were the most altered pathways in 22%, 16%, 12%, 10%, 9% and 9% of the CRC patients, respectively. These results would contribute to a better understanding of the colorectal cancer and in introducing personalized therapies for Egyptian CRC patients.This study aims at identifying common pathogenic somatic mutations at different stages of colorectal carcinogenesis in Egyptian patients. Our cohort included colonoscopic biopsies collected from 120 patients: 20 biopsies from patients with inflammatory bowel disease, 38 from colonic polyp patients, and 62 from patients with colorectal cancer. On top of this, the cohort included 20 biopsies from patients with non-specific mild to moderated colitis. Targeted DNA sequencing using a customized gene panel of 96 colorectal related genes running on the Ion Torrent NGS technology was used to process the samples. Our results revealed that 69% of all cases harbored at least one somatic mutation. Fifty-seven genes were found to carry 232 somatic non-synonymous variants. The most frequently pathogenic somatic mutations were localized in  Its rank jumped in females, however, to the fifth place  [Colorectal cancer (CRC) is one of the leading causes of mortality and morbidity worldwide. It is the third most common neoplasm and the second leading cause of cancer-related death worldwide . In Egypin 2015) . It is eNext generation sequencing (NGS) technology is an efficient means to characterize mutations associated with the disease in the patients’ genomes . It is fA number of studies have been published to characterize the somatic mutations and related genes associated with CRC worldwide ,5,6,7. In = 120) were collected from the patients classified into (1) inflammatory bowel disease , (2) colonic polyp  and (3) colorectal carcinoma patients  as well as extra participants with chronic non-specific mild to moderate colitis without any colonoscopic abnormalities . The collected colonoscopic biopsies were stored in MACS Tissue Storage Solution at −80° freezer until DNA extraction. Before the study began, each patient who was enrolled signed a written informed consent form. The Institutional Review Board of the National Cancer Institute (NCI), Cairo University, Egypt has authorized all human subject protocols and procedures .Fresh colonoscopic biopsy samples , and they are summarized in Our laboratory uses the Ion Torrent  sequencing technologies. The first version of the ready-to-use colorectal cancer gene panel kit based on this technology was the Ion AmpliSeq Colon/Lung panel. This panel appeared a few years ago and was composed of 22 genes. The recent version of this panel from Thermo Fischer is the Ion Torrent Oncomine Colorectal and Pancreatic Panel, composed of 24 genes . For our study, we wanted to have a disease focused panel, but with more genes specific to colorectal cancer. To this end, we studied the gene lists available in the commercial cancer gene panels, such as the Comprehensive Cancer Panel  composed of 409 genes, the different versions of the Oncomine Comprehensive panels , and the Qiagen’s GeneRead colorectal panel, composed of 38 genes. This is in addition to studying gene lists from literature, TCGA, and different commercial tests registered in the Gene Test Registry. Our final list included 96 genes, and it is given in ® DNA mini kit  according to the manufacturer’s instructions. The purified DNA was measured using Qubit® 3.0 Fluorometer  with Qubit™ dsDNA HS assay kit .The DNA was isolated from the collected biopsies using the QIAampThe QIAseq Targeted DNA technology from Qiagen was used to develop kits for our customized gene panel composed of the selected 96 genes . The NGS libraries were constructed according to the manufacturer’s instructions. After library preparation, the QIAxcel  was used to check the fragment size and concentration with the QIAxcel DNA high resolution kit . The prepared libraries were quantified using QIAseq Library Quant Assay Kit . Then, the libraries with different sample indexes were combined in equimolar amounts to achieve a similar sequencing depth for each combined library. The fragment size distribution in our libraries ranged between 200 and 1000 bp. The Ion PI Hi-Q Chef Kit , running on the Ion Chef, was used to load the combined libraries on the Ion PI Chip . The Ion Proton Platform was used for next generation sequencing using the Ion Proton Sequencing 200 Kit v2 .http://www.webgestalt.org (accessed on 3 November 2021)), Ingenuity Variant/Pathway Analysis , and web Reactome (https:/reactome.org (accessed on 15 November 2021)). For the alignment step, we used the human reference genome version hg19. The sequencing of a sample was accepted if the depth was larger than 100× and the coverage was more than 95% of the target regions.The Ion Torrent Suite was used for base calling, alignment, and variant analysis. First, the low-quality reads were excluded and the bases with base quality less than Q30 were trimmed. The alignment and variant calling proceeded in two parallel directions to identify the somatic variants: In the first direction, we used the somatic workflow in the Torrent Suite  to call the somatic variants. In the second, we used the QIAGEN GeneGlobe Data Analysis Center. Then, the variant lists from the two platforms were combined together and submitted to the annotation workflow. For annotation, we used QIAGEN GeneGlobe Data Analysis Center, Ingenuity Variant Analysis  , and theSomatic mutations in the cancer patients were identified based on the following multi-step filtering process introduced in . First, 2 test or Fisher’s exact test, when appropriate for the categorical variables, and by Mann–Whitney test or Student’s t-test when appropriate for the continuous variables. p-value was considered significant when p-value ≤ 0.05.The clinic-pathological features of the assessed patients were analyzed using SPSS software package (version 22). Continuous variables were expressed as mean ± SD and range, while categorical variables were expressed as percentages. Comparisons between groups were analyzed by χp-value < 0.001). Nearly half of the CP group (47%) had atypical lesions. The most predominate grade was grade II, found in 64% of the CRC group. Most of the CRC patients presented with non-metastatic and non-recurrent status   .In total, there were 232 somatic non-synonymous variants (73% SNPs and 27% Indels). The mean depth of coverage of the non-synonymous variants ranged from 500 to 1000× in all studied groups . Most ofTP53 , APC , KRAS , PIK3CA , POLE , MSH6 , FBXW7 , SMAD4 , ATM , and FGFR3 . In the CRC group, TP53 , APC , KRAS , PIK3CA , MSH6 , FBXW7 , SMAD4 , ATM , FGFR3 , BRAF , and POLE  were the top mutated genes. In the CP group, APC , TP53 , BRAF , FGFR3 , KRAS , and POLE  were the most mutated genes, while TP53 , APC , and POLE  were the most mutated ones in the IBD group.Fifty-seven genes out of the studied genes were found to carry somatic mutations . The topTP53, APC, and KRAS genes harbored the most frequently detected somatic mutations  in the total cohort. In the CRC group, TP53, APC, PIK3CA, KRAS, and ATM genes harbored the most frequently detected somatic mutations . In the CP group, APC, TP53, BRAF, FGFR3, and KRAS genes had the most frequently detected variants . Regarding the IBD group, TP53 and APC genes harbored the most frequent somatic mutations (five and four). The somatic mutational burden per sample in the CRC group was the highest, and it was about three variants per sample on average; this is followed by the CP (2.7) and the IBD groups (2.4). As for the somatic mutations that were detected in the top genes in each group, TP53 and APC genes were found to be the most affected genes, with somatic mutations in all groups, a schematic representation of their somatic mutations at protein level was made, as shown in TP53 harbored 36 mutations from 31 samples  and APC harbored 30 mutations from 26 samples . We found that exons 3 and 4 of the TP53 gene (NM_001126115) possessed a high number of mutations (28% and 25% respectively). In APC (NM_001127511), exon 14 harbors most of the mutations (85%). The β-catenin binding and down-regulation site was the most affected region at the APC protein, whereas the transactivation and the proline rich sites were the most affected regions at the TP53 protein.As the TP53 and APC are the genes with the greatest number of mutations . This is followed by the KRAS gene with two mutations, as shown in There are, in total, 16 somatic mutations detected in the CRC group and in either the IBD or the CP group: 10 SNPs and 6 Indels. APC, TP53, PIK3CA, FBXW7, ATM, and SMAD4, and they were housing 12, 11, 7, 6, 5, and 5 mutations, respectively. CRC-specific variants harbored by the top genes were reported in 23 out of total 35 CRC patients carrying CRC-specific mutations. Average CRC-specific variants per sample is 2.4, as shown in Eighty-four somatic variants harbored by 36 genes were found only in the CRC group, representing 51% of total somatic variants. The top genes carrying CRC-specific mutations are PIK3CA (NM_006218) and four in FBXW7 (NM_001013415), as shown in PIK3CA and FBXW7 mutations were reported in seven and four cases, respectively. Out of these 10 novel mutations, 8 mutations were found in the CRC group only, while a mutation in PIK3CA (c.1013T>A) is found in CP group only and a single mutation in FBXW7 (c.248C>T) is found in IBD group. The ten novel mutations have not been previously reported in any of the public databases.Moreover, ten novel heterozygous mutations were identified, including six in p-value = 1.28 × 10−08), Wnt signaling pathway (p = 0.0028), Angiogenesis , EGF-receptor , TGF-beta signaling , and Interleukin signaling . The most altered pathways in CRC patients and the distribution of the mutations of cancer driver genes are in Pathway analysis revealed that the following pathways were strongly suggested to be altered in the CRC group: P53-signaling pathway  were exon 3 and 4. In accordance with a recent study by Kassem et al. [TP53 somatic mutations c.628C>T, c.448C>T, c.347G>A, and c.128G>A in our cohort are specific to the CRC group, which suggests that they play a key role in the CRC in the Egyptian population. Additionally, we detected two TP53 variants (c.164C>G and c.137delC) that were previously reported in the esophageal and lung cancers. Interestingly, and according to the COSMIC database, this is the first study to report the presence of such variants in CRC [Moreover, eleven m et al.  on the Es in CRC ,24,25,26ATM gene, as a DNA repair gene, occur in many tumor types including colorectal cancer. In colorectal cancer, the loss of ATM protein expression is associated with worse prognosis [ATM gene was mutated in 12% of the CRC cohort; five out of the seven detected somatic mutations were found only in the CRC group. All of the observed ATM mutations had previously been linked to CRC [Somatic mutations of the rognosis . Therefod to CRC , with thd to CRC ,16,17,18ATM mutational status could be used to help in the clinical decision-making for those patients along with the development of specific targeted strategies [Nowadays, novel therapies have been developed to selectively target patients with ATM-deficient cancers. Those therapies induce synthetic lethality due to lacking an efficient repair mechanism such as platinum drugs . Thus, trategies . Thus, iAPC gene, a multi-functional tumor-suppressor gene, is an early event in the development of CRC and result in activation of Wnt/β-catenin signaling pathway, which is a key event for epithelial development [APC, Axin2, and AMER1 (APC-recruitment protein) disrupt the formation of the β-catenin destruction complex leading to stabilization and accumulation of β-catenin protein, which in turn induces overactivation of Wnt/β-catenin signaling and promotes the proliferation, invasion, and metastasis of cancerous cells [APC gene (NM_001127511) was the second ranked mutated gene (23% of the CRC cohort). There were 12 APC somatic mutations with identified loss of function detected only in the CRC group. Interestingly, exon 14 was the most affected exon and it was found to harbor 11 out of 12 detected mutations in the CRC group only. Thus, sequencing this exon could be used as a genetic test assay for CRC diagnosis. Of interest, most of our identified APC somatic mutations were located in the β-catenin binding and down-regulation site, which may result in an altered Wnt/βcatenin pathway. Meanwhile, the somatic mutations detected in AXIN2 (two mutations) and FZD3 (one mutation) were reported only in CRC patients, and they were participating in Wnt/βcatenin pathway as well. The two AXIN2 mutations (c.2347G>T and c.1102G>A) were previously addressed as being associated with small cell lung and prostate cancers, respectively [FZD3 mutation (c.674dupT) was previously reported in oral squamous cell carcinoma (OSCC) [Mutation of the elopment . Mutant us cells ,60. We hectively ,30, whila (OSCC) . TherefoAPC bi-allelic mutations, which in turn decrease the β-catenin degradation [CTNNB1 mutations are considered an alternative method for activation of Wnt signaling pathway [APC and CTNNB1 mutations is infrequent [CTNNB1 gene. It is another protein that acts as a gene expression regulator for Wnt downstream genes which is responsible for cell proliferation and differentiation [CTNNB1 gene are rare. Here in our study, we identified one CTNNB1 (NM_001098209) pathogenic variant in CRC patients and it was located on exon 10 (c.1561C>T). This was in agreement with Giannakis et al., who had detected the same mutation in one CRC patient, as well [The Wnt signaling cascade is mostly activated by radation . However pathway . Furtherfrequent . Beta-cantiation ,65. Besintiation ,67,68,69 as well .KRAS, NRAS, BRAF and PIK3CA) can determine the response to this target therapy. Hence, they can be used as predictive biomarkers. The following mutations were previously known to contribute to the acquired resistance to anti-EGFR target therapy, mutations in KRAS  [NRAS , BRAF (exon 15), and PIK3CA (exon 20) [PIK3CA and KRAS are often co-mutated and therefore can predict the lack of response to anti-EGFR therapy [Epidermal growth factor receptor (EGFR) is a trans-membrane protein. Bad prognosis and drug resistance in CRC is usually associated with EGFR overexpression ,72. The , and 4) ,74,75, Nexon 20) . PIK3CA  therapy ,78.KRAS and NRAS are members of RAS gene family. KRAS is one of the most frequently mutated genes, with alterations observed in 30–40% of CRC patients. On activation of KRAS, a signal transduction cascade will be initiated that will eventually promote many cell processes  through the subsequent activation of several target effectors  [NRAS mutations are identified in ~4% of CRCs [KRAS and NRAS mutations have less favorable prognoses, shorter survival, and increased tumor aggressiveness [KRAS and NRAS mutations in CRC predict lack of response to anti-EGFR MoAbs therapy [KRAS and NRAS mutations among our CRC patients were detected. In CRC patients, three were found to carry KRAS mutations in codon 13 (c.38G>A) while five CRCs carried KRAS mutations in codon 12  and both codons are located on exon 2. As for the NRAS mutations, one mutation in codon 12 on exon 2 (c.35G>T) was identified in one CRC patient only.In light of this, the genes implicated in the EGFR-signaling pathway will be discussed in the following lines.  PIK3CA) ,80. On t of CRCs . Additiosiveness . Moreove therapy ,83,84. IBRAF) which encompasses 18 exons. This protein is associated with the MAPK pathway, which is involved in carcinogenesis of many cancers [BRAF mutation leads to a constitutive activation of the MAPK signaling pathway, eliciting cellular proliferation, angiogenesis, and differentiation [BRAF alterations have been identified [BRAF mutations were observed in less than 10% of CRC. BRAF mutations may indicate an initial event in tumorigenesis [BRAF mutation is the V600E subtype  [BRAF 10 times relative to the BRAF wild type, which in turn promotes cell survival via the ERK or MEK signaling cascade [BRAFV600E inhibitors in CRC [BRAFV600 mutations in CRC patients  and these findings were also in agreement with Yanus et al. and Won et al. [Serine or threonine protein kinase is encoded by a proto-oncogene  . This mu cascade . Our dat cascade . To dates in CRC . Most ofs in CRC ,97. Heren et al. ,99.PIK3CA mutations are frequently associated with other gene mutations which are involved in significant cancer-related pathways, such as the Wnt/beta-catenin pathway and tyrosine kinase receptors K-Ras/BRAF/MAPK. The PIK3CA gene encodes the alpha catalytic subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), which is mutated in several malignancies  [PIK3CA mutations have been detected in 10 to 32% of colorectal tumors [PIK3CA somatic variants; out of these, five of them were likely to be novel , and they were located on exons 2, 2, 2, 21, and 20, respectively. In addition, our results are in agreement with Samuels et al., who has reported an association between PIK3CA mutation  and CRC [PIK3CA variant  detected in our CRC patients and colorectal cancer. This variant was previously addressed with lung cancer [KRAS, BRAF, and PIK3CA mutations, which was consistent with an Egyptian study by Farghal et al. [and CRC) . PIK3CA l tumors ,102. In  and CRC . Our stug cancer . FurtherKRAS located on exon 2, one mutation in NRAS located on exon 2, one BRAFV600, two non-BRAFV600 mutations located on exon 15, and one PIK3CA mutation located on exon 20. Thus, these data may provide beneficial information that helps in the clinical management regarding anti-EGFR therapy using a personalized medicine approach for the colorectal cancer patients in Egypt. In addition, we detected other mutations in CRC patients that are known to participate in the EGFR-signaling pathway, such as ERBB2 , and EGFR (c.2116C>T and c.630G>A).Matching with what we have mentioned before, we have detected somatic mutations in eleven CRC patients (18%) that are linked to the resistance to anti-EGFR target therapy, three variants in TP53 and ATM genes. Up-regulation of the Wnt/βcatenin pathway in CRC patients due to mutations in APC, AXIN2, and FZD3 reveal that the Wnt/βcatenin pathway plays a major role in sporadic colorectal carcinogenesis. Therefore, the dysregulation of both pathways in our CRC group may arise as an attractive therapeutic target [RAS gene can lead to PI3K/AKT/mTOR pathway activation that will promote the expression of other angiogenesis-associated factors  [AKT mutations [APC, CTNNB1, NRAS, PIK3CA, AKT2, and BRAF genes in our CRC patients, the dysregulation of the Angiogenesis pathway could be attributed to the interaction between Wnt/β-catenin, PI3K/AKT/mTOR, and MAPK signaling pathways, and this is in agreement with Lee et al. and Jeong et al. [When we performed pathway analysis, it revealed dysregulations of six pathways. Inactivation of P53 signaling pathway was detected in CRC patients due to the presence of deleterious mutations in c target ,106. Meac target . On actic target . In addioietins) . Angiogeoietins) . Moreoveutations . In the g et al. ,112,113.g et al. .SMAD4 mutations with the CRC, we detected five somatic mutations only in the CRC group [SMAD4 gene acts as an intracellular mediator of TGF-β superfamily signals. TGF-β/SMAD4 signaling maintains DNA damage response (DDR) and DNA damage repair [SMAD4 promotes malignant progression via acquiring resistance to TGF-β superfamily growth inhibition [SMAD4 occurs in the CRC in frequencies ranging from 9 to 67% [SMAD4 loss was also associated with worse clinical outcome and resistance to fluoropyrimidine-based chemotherapy [SMAD4 mutations may not benefit from fluoropyrimidine-based treatment.Matching with two studies that reported the association of RC group . The SMAe repair . Additioe repair . In thishibition . Moreovehibition . Isaksso9 to 67% . Moreoveotherapy , implicaotherapy . Thus, wEP300 gene has been previously observed in gastric, breast, pancreatic, and colorectal cancers. In addition, Gayther et al. reported a great relevance of the EP300 loss in colorectal carcinogenesis [EP300 gene harbored two missense mutations in CRC patients (c.1058G>A and c.3671+1G>A). They were previously detected in breast and gastric cancers, respectively [EP300 overexpression was an indicator of good prognosis in the CRC patients [EP300 gene might serve as a predictor of bad prognosis in Egyptian CRC patients. One of the most frequently detected somatic mutations in the CRC is in the tumor suppressor FBXW7 gene. Loss of the FBXW7 was reported to promote epithelial–mesenchymal transition (EMT) and metastasis in the CRC cells [FBXW7 gene that were found associated with CRC: two were previously reported with CRC patients , a single mutation was previously reported with breast cancer but was not addressed with CRC before (c.167A>G), and another three likely novel mutations  [The ogenesis . Our stuectively ,35. Morepatients . TherefoRC cells . The pre1030T>C) .ARIDA1 gene has been previously reported as a frequent event in the colorectal carcinogenesis [ARIDA1 mutations in CRC patients (only a single case in the CRC group). ACVR2A (activin A receptor type 2A) has a mutation rate of about 60%, making it the most frequently mutated gene in hypermutated colon cancer [The functional loss of the tumor suppressor ogenesis ,126. Hown cancer . It medin cancer . Here, wTP53), (c.38G>A in KRAS), and (c.4337_4338del in POLE). We found no significant increase in frequency of those mutations from IBD to, finally, CRC. Thus, we recommend increasing sample size to validate the association of these variants with disease progression in further studies.Regarding the common somatic mutations detected in all the studied groups, we have found that four mutations were the most frequent (c.137delC and c.422G>A in APC, TP53, PIK3CA, FBXW7, ATM, and SMAD4 were the top genes carrying CRC-specific mutations. APC and TP53 genes were the most affected genes in all groups; most of their deleterious somatic mutations were located in exon 14 and exon 3, respectively. Twenty-nine somatic mutations in 21 genes were found to be associated with CRC patients exclusively. Additionally, ten likely novel variants in PIK3CA and FBXW7 were identified in Egyptian CRC patients. P53, Wnt/βcatenin, Angiogenesis, EGFR, TGF-β, and Interleukin signaling pathways were found to be the most altered pathways in CRC patients. Furthermore, our findings revealed that 18% of CRC patients had somatic mutations linked to resistance to anti-EGFR target therapy, implying that 82% of patients could benefit from this treatment. This study may provide a better understanding of colorectal cancer and identification of cancer driver genes with cancer-specific variants in our patients, and these findings may assist in the development of diagnosis and novel personalized treatment regimens suited to Egyptian colorectal cancer patients. It is worth noting that our findings are confined to grade II patients because they account for 64% of all CRC cases.To the best of our knowledge, this is the first study on the sequencing of a multiple-gene panel for disease progression of Egyptian CRC patients. Our results revealed that