M. oleifera leaves extract increased by incorporating silver nanoparticles (Ag-NPs) then studied against colon cancer induced by azoxymethane (AOM) in rats. Colorectal cancer (CRC) categorized as the most common type of gastrointestinal cancers affected both genders equally. Chemotherapeutic drugs became limited due to their deleterious side effects. Therefore, efficiency of TP53) and Adenomatous Polyposis Coli (APC) genes in colonic tissues.Different hematological and biochemical measurements in addition to specific tumor and inflammatory markers were quantified. Histopathological examination for Colonic tissues was performed. Native proteins and isoenzyme patterns were electrophoretically detected in addition to assaying expression of Tumor Protein P53  values were noticed with electrophoretic protein (SI=61.54%), lipid (SI=0.00%) and calcium (SI=75.00%) moieties of protein patterns, catalase (SI=85.71%), peroxidase (SI=85.71%), α-esterase (SI=50.00%) and β-esterase (SI=50.00%) isoenzymes in addition to altering the relative quantities of total protein and isoenzyme bands in colon of cancer induced group. Moreover, levels of TP53 and APC gene expression increased significantly (P≤0.05) in colon cancer induced group. The nano-extract prevented the qualitative and quantitative alterations in the different electrophoretic patterns in addition to restoring levels of the gene expressions to normalcy in both of simult- and post-treated groups. M. oleifera nano-extract exhibited ameliorative effect against the biochemical, physiological and molecular alterations induced by AOM in nano-extract simult- and post-treated groups. Cancer categorized as one of the leading causes of death worldwide. Behaviors of the lifestyle including smoking, poor diet, physical inactivity and reproductive changes increase susceptibility of the body to cancer incidence in less economically developed countries . It is largely occurred as a result of one or more of the genetic mutations that might be inherited or acquired . Shepherd and Sridhar (2003) postulated that cancer development is a complex multistage process, involving not only genetic change, but also due to hormonal changes, cocarcinogen and tumor promoter effects such as activation of proto oncogenes to oncogenes and inactivation of tumor suppressor genes.Based on Globocan estimates, it was reported that colorectal cancer (CRC) is the third most lethal cancer affecting both genders equally in developed countries . During the CRC pathogenesis, sequential and multistep progression of epithelial cells initiated to a cancerous state with defined precancerous intermediaries . At different stages of carcinogenesis, the specific oncogenes and tumor suppressor genes altered and this consequently leads to accelerating the cancer incidence . There are two or three substances suitable to induce colon tumors. CRC induced chemically as a result of azoxymethan (AOM) injection within 7 - 9 months . The induction process accelerated after administrating an inflammatory agent in drinking water to murine models following AOM injection. Dextran sodium sulfate (DSS) is the most effective inflammatory agent suitable to accelerate CRC induction within a short period . Carbohydrate antigen (CA) 19-9 and carcinoembryonic antigen (CEA) used as indicators for a biological state of colon tissues and their levels are related to CRC progression . Therefore, they can be used for early CRC diagnosis. The tumor suppressor genes responsible for expression of Tumor Protein P53 and Adenomatous Polyposis Coli (APC) are susceptible to the genetic mutations that play an important role in CRC progression .M. oleifera) Lam.  belongs to the Moringaceae family . M. oleifera leaves are considered as a potential source of active phyto-constituents such as total phenolics, vitamins , ascorbic acid oxidase, polyphenol oxidase and catalase in addition to the minerals . It was found that these constituents exhibit antioxidant and scavenging activities against oxidative stress induced by reactive oxygen species (ROS) . M. oleifera leaves are rich in high protein content that is considered as an ideal source of essential amino acids such as methionine, cysteine, tryptophan and lysine. They exhibited good availability for intestinal absorption and rumen degradability of nitrogen as compared to soybean meal .Chemotherapy, radiotherapy, surgery, hormonal and targeted therapy belong to the modalities for cancer treatment. Most of these available therapeutic options have different side effects . Cancer treatment by mean of chemotherapy became limited due to the deleterious side effects and multidrug resistance . As reported earlier by Craig and Beck (1999), about 74% of known anti-cancer agents that currently used nowadays were derived from various plant species. Moringa oleifera , anti-atherosclerotic , anti-fertility, anti-depressant  and diuretic effects . Moreover, administration of M. oleifera extracts alone lead to significant increase in RBCs and Hb as compared to control group. Therefore, M. oleifera extract exhibited beneficial effect against mitigating anemia induced chemically in rats . Furthermore, the plant extracts attenuated brain dysfunction and brain damage . It ameliorated the brain damage and toxicity of the cerebral cortex induced by lead injection . The studies focused recently on evaluation of M. oleifera extract efficacy with respect to tumor-suppressive activity, but not on the molecular basis of the tumor-suppressive activity .Many reports have described the potential therapeutic values of Synthesis of metal nanoparticles (M-NPs) attracted the attention due to their different characteristics as compared to macrostructures . Synthesis of silver nanoparticles (Ag-NPs) by reduction of aqueous silver nitrate into Ag-NPs during the exposure to plant extracts can be easily monitored by using UV-visible spectrophotometer. The plant extracts with Ag-NPs exhibited good antioxidant activity at lower concentrations . Recently, it was found that incorporation of Ag-NPs in the plant extract increased the total phenolic compounds and total flavonoids. Therefore, silver plant nano-extracts showed a higher antioxidant and antimicrobial activity compared to plant extract alone or silver nitrate .M. oleifera leaves extract to enhance its antioxidant efficiency as suggested by Shousha et al., (2019) and supported by Aboulthana et al., (2021) then to evaluate nano-extract against CRC induced by AOM in rats. Most of the biologically active components absorbed slowly due to their high molecular weights which decreases their ability to cross the cellular membrane and hence decreases their efficacy and bioavailability. Therefore, the traditional medicinal plants integrated with nanotechnology to solve the stability problems for increasing their bioavailability and reducing their toxicity . Development of metal plant nano-extracts showed great promise to solve their inherent stability problem . Incorporation of M-NPs into plant extracts increased the biological activities at lower concentrations as compared to plant extract solely and hence it increases the practical applications . From this point of view, the study was designed to develop novel strategy for CRC treatment by mean of green nanotechnology through incorporating Ag-NPs into Silver M. oleifera Leaves Nano-extract AdministrationM. oleifera nano-extract was prepared and characterized by advanced confirmatory technique and administrated orally by stomach tube at a dose of 687.50 mg/Kg body weight (b.wt) (1/20 of LD50).Based on our study published recently , the aqueous silver Induction of Colon Cancer in RatsM. oleifera nano-extract against induced colon cancer. Rats were injected by a single injection of AOM at a dose of 20 mg/kg b.wt interperitoneally (i.p.) to induce colon cancer then rats were given 2% DSS in drinking water for 7 days after one week of AOM injection . Induction of the disease was detected starting from 8th week of AOM injection by histological examination. It was noticed that the disease induced after 12 weeks of AOM injection. Consequently, the induced rats were used to evaluate efficacy of the prepared silver Experimental Design Healthy thirty-six adult male Wistar rats (weighting 120 - 150 g) were divided into six groups and housed in 6 cages (six per cage). Rats were provided with water ad libitum and maintained under normal nutritional and environmental conditions at 25 ± 2 °C. The experimental procedures were carried out based on the ethical protocol and guidelines approved by the institutional animal care of National Research Centre, Dokki, Giza, Egypt. M. oleifera nano-extract treated group: Rats were fed with normal diet associated with the treatment with M. oleifera nano-extract orally at a dose of 687.50 mg/Kg b.w. for 21 days, Colon cancer induced group: Rats were injected with AOM at a dose of 20 mg/kg i.p., Silver M. oleifera nano-extract pre-treated group: Rats were treated with M. oleifera nano-extract orally at a dose of 687.50 mg/Kg b.w. for 21 days followed by injecting AOM i.p. for another 12 weeks, Silver M. oleifera nano-extract simult.-treated group: Rats treated simultaneously with AOM i.p. and treated orally by M. oleifera nano-extract and Silver M. oleifera nano-extract post-treated group: Rats were injected AOM i.p. for 12 weeks followed by the treatment with M. oleifera nano-extract orally at a dose of 687.50 mg/Kg b.w. for another 21 days.Rats were randomly divided into Control group: Rats were fed with normal diet as ad libitum and received distilled water for 21 days, Silver Collection of Blood Samples and TissuesAt end of the experimental period, rats were anaesthetized then the heparinized blood samples were collected from the retro orbital plexus for the hematological measurements. For biochemical measurements, the sera were collected by centrifuging blood samples at 4,000 rpm for 15 min. After sacrificing by decapitation, colon tissues were excised and washed in ice-cold saline. Each colon tissue was splitted into 2 portions. Portion (I) was immediately fixed in formal saline (10%) for histopathological examination. Portion (II) was immediately washed in physiological saline then homogenized in Tris-HCl buffer (0.01 M and pH 7.4). Aliquots of the clear supernatants were used for quantifying markers of the inflammatory response and oxidative stress and for assaying the native electrophoretic patterns.Hematological and Biochemical MeasurementsThe hematological measurements including parameters of the complete blood count were quantified in heparinized blood samples using an automatic blood analyzer (ABX Micros 60 manufactured by HORIBA ABX SAS). The biochemical parameters  and aspartate transaminase (AST), lipid  and triglycerides (T.Gs)), renal functions (urea and creatinin) and protein measurements ) were estimated in serum samples by colorimetric methods using the commercially available kits purchased from Spectrum Diagnostics Egyptian Company for Biotechnology .Tumor MarkersThe specific tumor markers (Carcinoembryonic Antigen (CEA) and Carbohydrate Antigen (CA) 19-9) that were released in case of colon cancer incidence were quantitatively assayed in sera of all treated groups using Enzyme Linked Immune Sorbent Assay (ELISA) technique.Inflammatory MarkersThe inflammatory markers that were expressed in response to incidence of colon cancer include C reactive protein (CRP) level and Myeloperoxidase (MPO) activity. The CRP level was determined in sera specimens and expressed as mg/L using commercially available enzymatic kit . The MPO activity was quantified in supernatant of colon tissue homogenates and expressed by Δ/min/mg protein according to the method suggested by Correia et al., (2012).The Major Cellular Contents in Colon Tissues HomogenatesIn colon tissues homogenates, calcium concentration was determined using fully automated with microprocessor-controlled electrolyte system (Sensa Core’s ST-200 CL). Activity of lactate dehydrogenase (LDH) was measured according to the method described by Babson and Babson (1973). Glucose level was measured by enzymatic colorimetric method using commercial Kit .Markers of Oxidative StressThese markers were assayed in clear supernatants of colon tissue homogenates. Lipid peroxidation product (LPO) was measured as malondialdehyde (MDA) which is the end product of peroxidation reaction and the result was expressed as nmol/g wet tissue . Total protein carbonyl content (TPC) was quantified according to the method suggested by Levine et al. (1994) and the result was expressed as nmol of reactive carbonyl compounds per mg protein of tissue. Total antioxidant capacity (TAC), catalase (CAT) and glutathione peroxidase (GPx) activity were assayed according to the methods suggested by Koracevic et al. (2001) as mM/L, Aebi (1984) and Paglia and Valentine (1967) as Unit per gram of tissue, respectively.Statistical Analysis Data compiled in tables and illustrated figures were expressed as mean ± standard error (SE). They were analyzed statistically by one-way analysis of variance test (one-way ANOVA) using the Statistical Package for Social Sciences  followed by Bonferroni test as Post-Hoc. The difference between the groups at a “P” value of less than 0.05 was considered statistically significant.Histopathological ExaminationSpecimens were autopsied from colonic tissues of different groups and immediately fixed in formal saline then studied histopathologically according to the protocol described by Banchroft et al., (1996). The washed tissue specimens were dehydrated in serial alcohol solutions and cleared in xylene then embedded in paraffin wax blocks. The tissue sections (at thickness 4 microns) were collected on glass slides and deparaffinized then stained by hematoxylin & eosin (H&E) stain to be examined under the light electric microscope. The histopathological changes were assigned and scored then rated between 0 (no damage) and +++  .Electrophoretic AssaysKnown weights of colon tissues (0.2 gm) were freezed rapidly in liquid nitrogen then homogenized in 1 ml water-soluble extraction buffer. The homogenates were centrifuged for 5 min at 10,000 rpm then the clear supernatants separated and transferred into another tubes. Equal volumes of the individual supernatants in each group were pooled together and used as one sample. Total protein concentration was measured in all pooled samples according to the method suggested by Bradford (1976) to ensure that concentration of the protein loaded in all wells equal during all electrophoretic assays. Native proteins were separated electrophoretically through Polyacrylamide Gel Electrophoresis (PAGE) then stained by commassie brilliant blue according to the method documented by Laemmli (1970) and improved recently by Darwesh et al., (2015). After electrophoretic run, native gels were stained for detecting lipid moiety of native protein by Sudan Black B (SBB) and the stained lipoprotein bands appeared as black bands . For detecting calcium moiety of native protein, native gels were stained using alizarin Red “S”. The stained calcium moieties appeared as yellow bands . For detecting the electrophoretic isoenzymes, electrophoretic catalase (CAT) and peroxidase (POX) patterns were assayed after electrophoretic run by incubating native gel with H2O2 as substrate then stained. The stained CAT subunits appeared as yellow bands  and the stained POX subunits appeared as brown bands in the gel . The electrophoretic esterase (EST) pattern was carried out to localize in-gel EST activity by incubating native gel in conditioning buffer then staining in reaction mixture consisting of α, β-naphthyl acetate  as substrates along with dye coupler Fast Blue RR. The α-naphthyl acetate used as substrate for detecting α-esterase (α-EST) that appeared as brown bands and β-naphthyl acetate used as substrate for β-esterases (β-EST) that appeared as dark pink bands .Data AnalysisThe photographed PAGE plates were analyzed using Quantity One software (Version 4.6.2). This program used for determining the relative mobility (Rf), band percent (B%) and relative band quantity (Qty%) of the electrophoretically separated bands in addition to the molecular weights (Mwts) that were determined as compared to marker of standard molecular weights with regularly spaced bands ranging from 6.458 to 195.755 KDa. The similarity index (SI%) and genetic distance (GD%) were calculated according to the equation suggested by Nei and Li (1979).Molecular AssayRNA Extraction and cDNA Synthesis® . Purity of the RNA yield was accepted in all samples (A260/A280 absorbance ratio of between 1.85 and 2.0). All RNA samples were normalized to 1,000 ng/μL, DNase treated  and reverse transcribed into First Strand cDNA using Power cDNA Synthesis Kit (iNTRON Biotechnology) according to manufacturers’ protocols.Total RNA was extracted from colon tissues using easy-spinTM Total RNA Extraction Kit (iNTRON Biotechnology). Concentration and purity of the RNA yields were measured using NanoDropPrimer Design Specific primers for APC  and tumor protein P53 (TP53)  genes were designed on the bases of the Rattus norvegicus sequence Gene bank accessions NM_012499 and NM_030989 respectively using Primer Quest Tool and synthesized by Macrogen company. The designed primers were used for Real time quantitative PCR (qRT-PCR). For gene expression analysis, Beta actin (Actb) was used as endogenous control and its primers  were designed based on sequence of Gene bank NM_031144.Gene Expression Using qRT-PCRAPC and TP53 genes were determined in colon tissues of control and different treated groups. A master mix was prepared for each assay containing 10μL of 2X SYBR green PCR mix , 1 ul of 10uM primer sets and nuclease-free water. For each sample, a 25 μL reaction volume of mastermix containing 1 ul of cDNA was prepared. The qPCR was performed by Qiagen’s real-time PCR system (Rotor-Gene Q) using default cycling conditions : 40 cycles of an initial activation step of 95°C for 15 min followed by 95°C for 10 s (melt), 57°C for 15s  and 72°C for 30s (extend). The mean Cycle threshold (Ct) values were calculated and then normalized to Actb using ΔCt method. Changes in relative expression were calculated using the 2-ΔΔCt method .Expression of the Statistical AnalysisMolecular data were statistically analyzed using the SPSS computer program . The non-parametric Mann-Whitney test used to compare levels of the relative gene expression in all treated groups to control. Results were reported as the arithmetic mean ± SE. The “P” value less than 0.05 is considered statistically significant .M. oleifera leaves extract after incorporating Ag-NPs against AOM induced colon cancer, it was found that AOM caused severe alterations at the hematological and biochemical levels during induction of the disease.Although the study was designed to reveal efficiency of Hematological MeasurementsAs depicted in Biochemical Measurements As reported in Tumor MarkersThe current study was concerned with assaying CEA and CA 19.9 that are considered as the most specific markers for colon cancer. It was found that levels of these tumor markers elevated significantly (P≤0.05) in AOM induced colon cancer group as compared to control group . The nanInflammation MarkersM. oleifera nano-extract caused significant (P≤0.05) decrease in levels of these inflammatory markers in all nano-extract treated groups as compared to colon cancer induced group, it restored levels of these measurements to normal values in nano-extract simult- and post-treated groups.As regard to the inflammatory response, CRP is considered as the most common marker of the inflammatory reactions. Also, MPO activity categorized as the most specific inflammatory marker expressed as a result of colon cancer induction. Data illustrated in Major Colonic Contents and Oxidative Stress MarkersAs regard to the assays that were carried out in supernatants of colon tissues homogenates, glucose and calcium in addition to activity of LDH enzyme are considered as the major cellular contents in colon tissues. Data recorded in Markers of the oxidative stress were represented by total antioxidant capacity (TAC) and activities of the antioxidant enzymes (CAT and GPx) in addition to products of the peroxidation reactions (LPO and TPC) that were assayed in colon tissues homogenates. Data recorded in Histopathological ExaminationM. oleifera nano-extract treated group . While in the other nano-extract treated groups, the nano-extract exhibited improvement in protein pattern by hiding the abnormal bands and restoring the normal ones  that were identified in nano-extract simult-treated group at Rfs 0.57, 0.63 and 0.78  and identified in post-treated group at Rfs 0.56, 0.63 and 0.77 . Therefore, the SI values increased and reached the highest value and hence the GD value decreased in that groups .From the SI% values, it was found that AOM induced colon cancer and nano-extract pre-treated groups were physiologically similar to control by 61.54%. Both of the nano-extract simult- and post-treated groups were qualitatively similar to control by 100%. AOM induced colon cancer group was similar to nano-extract pre-treated group by 100% and similar to the other nano-extract treated groups by 61.54%. As regard to the quantitative level, it was found that the relative quantities of the total protein bands declined significantly (P≤0.05) in colon cancer induced group as compared to control group . The nanElectrophoretic Lipid Moiety of Native Protein PatternAs revealed in The nano-extract exhibited improvement in lipid moiety of native protein pattern by restoring one normal band identified at Rf 0.08 (B% 49.03 and Qty% 14.01) in nano-extract pre-treated group with existence of one abnormal band at Rf 0.50 (B% 50.97 and Qty% 27.97). So, the SI value increased with decreasing the GD value  in that group. In the other nano-extract treated groups, the nano-extract exhibited physiological improvement through hiding the abnormal band with restoring the normal bands (2 bands) that were identified in nano-extract simult-treated group at Rfs 0.07 and 0.73  and identified in the post-treated group at Rfs 0.05 and 0.73 . Therefore, the SI value increased and reached the highest value and hence the GD value decreased in that groups .From the SI% values, it was found that there was no similarity (SI=0.00%) between AOM induced colon cancer group and control group. Both of the nano-extract simult- and post-treated groups were qualitatively similar to control by 100%. While the nano-extract pre-treated group was similar to control by 50%. There was no similarity (SI=0.00%) when colon cancer induced group compared to all nano-extract treated, groups. At quantitative level, the relative quantities of the total lipoprotein bands declined significantly (P≤0.05) in colon cancer induced group as compared to control group . The nanElectrophoretic Calcium Moiety of Native Protein PatternM. oleifera nano-extract alone caused no deviation detected in this protein pattern from the control group. AOM caused physiological alterations in calcium moieties of native protein pattern represented by appearance of 2 abnormal bands identified in colon cancer induced group at Rfs 0.34 and 0.89 . Therefore, the SI value decreased with increasing GD values in colon cancer induced group .Data illustrated in The nano-extract could not prevent or minimize the qualitative alterations induced by AOM in nano-extract pre-treated group. On the contrary, the changes became severe and represented by hiding normal band with existence of abnormal one identified at Rf 0.90 . Therefore, the lowest SI and highest GD values were recorded  in that group. In the other nano-extract treated groups, the nano-extract exhibited physiological improvement through hiding the abnormal bands  with restoring the normal one that identified in the nano-extract simult-treated group at Rf 0.65  and identified in post-treated group at Rf 0.65 . Therefore, the SI values increased and reached the highest value and hence the GD values reached the lowest in that groups .From the SI% values, AOM induced colon cancer and nano-extract pre-treated groups were physiologically similar to control by 75.00 and 66.67%, respectively. Both of the nano-extract simult- and post-treated groups were similar to control by 100%. The colon cancer induced group was similar to nano-extract pre-, simult- and post-treated groups by 75.00%. At the quantitative level, it was noticed that the relative quantities of the total bands increased significantly (P≤0.05) in colon cancer induced group as compared to control group . The nanElectrophoretic IsoenzymesElectrophoretic Catalase PatternM. oleifera nano-extract alone caused no abnormalities or deviation from control group. AOM caused physiological changes in the CAT pattern represented by hiding the CAT1 type without appearance of abnormal ones. Therefore, the SI decreased associated with increasing GD values in colon cancer induced group .As presented in The nano-extract could not prevent or minimize the qualitative alterations induced by AOM in the nano-extract pre-treated group. On the contrary, the changes became severe and represented by hiding the CAT1 type with existence of 2 abnormal (characteristic) bands identified at Rfs 0.34 and 0.67 . Therefore, the lowest SI and highest GD values were recorded in that group . In the other nano-extract treated groups, the nano-extract exhibited physiological improvement by restoring the CAT1 type  that identified in nano-extract simult-treated group at Rf 0.13  and identified in post-treated group at Rf 0.12 . Therefore, the SI values increased and reached the highest value with decreasing the GD values in that groups .From the SI% values, it was found that the induced colon cancer and nano-extract pre-treated groups were qualitatively similar to control by 85.71 and 66.67%, respectively. Both of the nano-extract simult- and post-treated groups were similar to control by 100%. The colon cancer induced group was similar to the nano-extract pre-, simult- and post-treated groups by 75.00, 85.71 and 85.71%, respectively. As regard to the quantitative level, it was found that the relative quantities of the total CAT types decreased significantly (P≤0.05) in colon cancer induced group as compared to control group . The nanElectrophoretic Peroxidase PatternAs revealed in The nano-extract could not prevent or minimize the qualitative alterations induced by AOM in the nano-extract pre-treated group. On the contrary, it increased severity of the abnormalities that represented by hiding the POX3 type with existence of one abnormal (characteristic) band identified at Rf 0.41 . Therefore, the lowest SI and highest GD values were recorded in that group . In the other nano-extract treated groups, the nano-extract exhibited physiological improvement by restoring the POX3 type  that identified in nano-extract simult-treated group at Rf 0.57  and identified in the post-treated group at Rf 0.57 . Therefore, the SI values increased and reached the highest value and hence the GD values reached the lowest values in that groups .From the SI% values, it was found that induced colon cancer and nano-extract pre-treated groups were qualitatively similar to control by 85.71 and 75.00%, respectively. Both of the nano-extract simult- and post-treated groups were similar to control by 100%. The induced colon cancer group was similar to nano-extract pre-, simult- and post-treated groups by 57.14, 85.71 and 85.71%, respectively. At the quantitative level, it was emphasized that the relative quantities of the total POX types decreased significantly (P≤0.05) in colon cancer induced group as compared to control group . The nanElectrophoretic Esterase PatternM. oleifera nano-extract alone caused no deviation from control group. No common bands were detected. Two characteristic (unique) bands were noticed. One of them was identified in colon cancer induced group at Rf 0.43 (B% 47.82 and Qty% 14.92) and the other identified in nano-extract pre-treated group at Rf 0.51 (B% 50.16 and Qty% 19.35). AOM caused qualitative abnormalities in the α-EST pattern represented by hiding the α-EST2 type. Therefore, the SI decreased associated with increasing GD values in colon cancer induced group .As illustrated in Nano-extract could not prevent or minimize the physiological alterations induced by AOM in the nano-extract pre-treated group. The changes were represented by hiding the α-EST1 type with existence of one abnormal (characteristic) band identified at Rf 0.51 (B% 50.16 and Qty% 19.35). Therefore, the SI% and GD% values were not changed as compared to colon cancer induced group . In the other nano-extract treated groups, the nano-extract exhibited qualitative improvement by restoring the normal α-EST2 type that identified in the nano-extract simult-treated group at Rf 0.27  and identified in the post-treated group at Rf 0.27 . This was associated with hiding the characteristic band. Therefore, the SI values increased and reached the highest value and hence the GD values reached the lowest values in that groups .From the SI% values, it was found that induced colon cancer and nano-extract pre-treated groups were qualitatively similar to control by 50.00%. Both of the nano-extract simult- and post-treated groups were similar to control by 100%. The induced colon cancer group was similar to the nano-extract simult- and post-treated groups by 50.00%. There was no similarity (SI=0.00%) between the colon cancer induced group and nano-extract pre-treated group. As regard to the quantitative level, it was found that AOM caused no significant changes in the relative quantities of the total α-EST types and values of the relative quantities of total α-EST types were approximately equal in all nano-extract treated groups as compared to control group and colon cancer induced group .As presented in The nano-extract could not ameliorate the qualitative alterations induced by AOM in the nano-extract pre-treated group. The changes were represented by hiding the β-EST1 type with existence of one abnormal (characteristic) band identified at Rf 0.81 (B% 47.53 and Qty% 32.02). The SI% and GD% values were not changed as compared to colon cancer induced group . In the other nano-extract treated groups, it improved the β-EST pattern by hiding the abnormal band with restoring the normal β-EST1 type that identified in the nano-extract simult-treated group at Rf 0.17  and identified in the post-treated group at Rf 0.15 . Therefore, the SI values increased and reached the highest value with decreasing the GD values in that groups . From the SI% values, it was found that the induced colon cancer and nano-extract pre-treated groups were qualitatively similar to control by 50.00%. Both of the nano-extract simult- and post-treated groups were similar to control by 100%. The colon cancer induced group was similar to the nano-extract pre-, simult- and post-treated groups by 100.00, 50.00 and 50.00%, respectively. There was complete similarity (SI=100.00%) between colon cancer induced group and nano-extract pre-treated group. At the quantitative level, it was fund that the relative quantities of the total β-EST types decreased significantly (P≤0.05) in colon cancer induced group as compared to control group. The nano-extract increased the relative quantities significantly (P≤0.05) in all nano-extract treated groups with respect to colon cancer induced group and restored quantities of the total β-EST types to normal values . Molecular assayTP53 gene expression as compared to control group. In addition, levels of TP53 gene expression were slightly significantly (P=0.026) higher in nano-extract pre-treated group as compared to control group. While in the other nano-extract treated groups, the simult- and post-treated groups didn’t show significant change in level of TP53 gene expression than control group .As illustrated in APC gene expression as compared to control group. In addition, levels of APC gene expression were significantly (P=0.037) higher in the nano-extract pre-treated group in comparison to control group. While the nano-extract simult- and post-treated groups didn’t show significant change in level of APC gene expression than control group .As revealed in Carcinogenesis is conventionally defined by three stages: initiation, promotion and progression. The normal cells experience DNA damage (initiation stage). The initiated cells affect normal cells forming preneoplastic lesions (promotion stage). The malignant tumors develop finally leading to metastasis (progression stage) . It is well known that the synthetic chemotherapeutic agents have the ability to modify the intracellular pathways to slow, block or reverse the carcinogenesis . The present study was designed to develop novel strategies for treatment of colon cancer by mean of green nanotechnology through incorporating Ag-NPs into plant extract to enhance its biological efficiency as suggested recently by Aboulthana et al., (2019).M. oleifera nano-extract restored levels of hematological measurements to normal values in the simult- and post-treated groups and this was in agreement with Dhar and Gupta (1982) who reported that leaves of this plant are rich in various vitamins and Fe in significant amount sufficient to improve the blood status of the body.The haematological parameters were assayed due to the effective role of immune system in controlling tumor development to reveal relation of blood constituents to colon cancer and for explaining the blood relating functions of plant extract . The haematological alterations occurred mainly as a result of AOM injection attributed to the oxidative stress and attack of the free radicals that closely related to cancer progression . During the present study, it was noticed that RDW level elevated significantly in colon cancer induced group. This agreed with Yang et al., (2018) who postulated that the RDW level is closely related to prolonged inflammatory processes in the active malignancy and metastasis of colorectal cancer. The MPV is an early marker of platelet activation that consequently plays a key role in inflammation and atherothrombosis. Its level elevated significantly in colon cancer induced group and this was in agreement with Li et al., (2014) who postulated that elevation on MPV level attributed to its positive correlation to stage of tumor-nodule-metastases. Also, the WBCs and their differential count  increased in colon cancer induced group due to stimulating the bone marrow through the inflammatory reactions to produce more leukocytes to migrate to inflammation site and this process was facilitated by PLT . M. oleifera nano-extract contains higher concentrations of the active ingredients that exhibit antioxidant activity when compared to the crude one. Consequently, improvement towards normalization of the biomarker enzymes following nano-extract administration suggested that the extract exhibited functions in preserving structural integrity of hepatocellular membrane against ROS generated as a result of AOM injection.Activities of the ALP, AST and ALP enzymes elevated significantly in colon cancer induced group and this agreed with Megaraj et al., (2014) who postulated that AOM induced hepatotoxicity and liver injury. This might refer to effect of the alkyl free radicals and the carbonium ions that belong to the highly reactive electrophiles targeting liver tissue and consequently leads to loss of membrane integrity and hence increasing membrane permeability. Therefore, these cytoplasmic enzymes leak at early stages of liver damage from hepatocytes into blood stream . The nano-extract restored activities of the liver enzymes to normal levels and this might be attributed to presence of wide range of the polyphenolic compounds that increased after incorporating Ag-NPs as supported by Shousha et al. (2019) who emphasized that M. oleifera nano-extract restored levels of the renal measurements to normal levels in all nano-extract treated groups. This was in accordance with Ameh and Alafi (2018) who reported that M. oleifera extract ameliorated the decline in protein content probably by scavenging ROS and improving the antioxidative status. Also, it might be attributed to presence of high concentrations of phenolic compounds that play an effective role in improvement of renal physiology and possess diuretic and natriuretic properties, as well as exerting the renoprotective effects that the antiapoptotic and antifibrotic properties of theses active constituents . In addition, the TP and albumin levels restored to normalcy due to the beneficial effect of M. oleifera nano-extract on liver tissues that became able to biosynthesize protein normally.Urea and creatinin were considered as indicators of kidney function and index of kidney dysfunction . In the present study, it was noticed that AOM caused renal toxicity represented by raising their concentrations and this might refer to effect of AOM that belongs to the hydrazine compounds that up-regulate biosynthesis of amino acids by stimulating urea cycle for removing excess ammonia derived from elevated amino acids . Moreover, urea level might be elevated as a result of converting amino groups (N-terminus) of amino acids into urea through urea cycle . Also, levels of the renal measurements might be increased with lowering TP and albumin levels due to the severe inflammatory and necrotic effects induced in kidney tissues by AOM injection . Levels of TP and albumin might be declined as a result of elevating levels of urea and creatinin that act as an inhibitor of protein synthesis  and / or the liver injury induced by AOM and consequently leads to the alterations in protein biosynthesis . Lipids are important components in the cell membranes for maintaining their architecture . T.Gs are the principal storage form of energy . In the present study, it was found that AOM caused significant elevation in levels of TC and T.Gs. This agreed with Yang et al., (2013) who showed that colorectal polyps were significantly associated with increased the TC and T.Gs levels. This might be attributed to various biochemical mechanisms proposed for the link between serum lipids and colorectal polyps. Moreover, the hypercholesterolemia is associated with the hyperinsulinemia and insulin resistance that can induce risk of the colorectal cancer . The carcinogenic substances that induce cancer cause defective lipids degradation or abnormal lipid synthesis. Also, the lipid measurements might be elevated due to deficiency of the lipoprotein lipase that stimulates the T.Gs uptake . Also, hypertriglyceridemia occurs due to the tumors growth and it is strongly associated with metastatic conditions . The nano-extract lowered levels of these lipid measurements due to efficiency of the active constituents in nano-extract to develop lipolysis-stimulating agent in adipocytes .CEA and CA19-9 exhibit their functions as a regulator for innate immune system, promoter for cellular aggregation and mediator for signal transduction. Therefore, they play an important role in tumor invasion and metastasis . Their levels are considered as the most common biomarker for CRC and characterized tumor-associated antigens in both biochemical and clinical aspects . Moreover, their levels are used for preoperative assessment of extent and outcome of cancer in addition to the postoperative monitoring of recurrence . In the present study, it was found levels of CEA and CA19-9 were elevated significantly in AOM induced colon cancer group. This agreed with Mare et al. (2013) who reported that AOM was categorized as a potent carcinogen that stimulates the oxidative stress and produces excess of ROS that cause metabolic instability in colon tissue leading to different changes in these tumor markers. Also, their levels were significantly correlated with lesion size and multiplicity of adenomas . The nano-extract decreased levels of these tumor markers and this might attributed to presence of the active ingredients and several secondary metabolites that are characterized by their antioxidant and anti-tumor activities . Their anti-tumor and antioxidant efficiency  increased after incorporating Ag-NPs .M. oleifera nano-extract decreased levels of the inflammatory markers and this in accordance with Tad and Dass (2019) who emphasized that the anti-inflammatory effect of the plant extract against intestinal inflammation might be attributed to presence of the secondary metabolites such as alkaloids, flavonoids, terpenoids and saponins that well known by their ability to inhibit the inflammatory reactions. Incorporation of Ag-NPs into plant ex¬tract showed higher antioxidant capacity. Correlation between total phenolic con¬tent and antioxidants capability showed the strong influence of phenolic compounds in the antioxidant activity of Ag-NPs and leaves extracts as well.The CRP is a positive protein in the acute phase of tissue damage and inflammation and elevation of its level reflected the ongoing inflammation and tissue damage much more accurately than other biochemical parameters . In the present study, AOM caused significant elevation in CRP level and this was in accordance with Rajendiran et al., (2018) who documented that CRP level in serum is significantly correlated to severity of colon inflammation and also this might be attributed to the inflammatory effect of the DSS that ingested via drinking water in combination with AOM during the induction process. Furthermore, the MPO activity is used as a good index of tissue injury induced by the inflammatory reactions . During the current study, the elevation in MPO activity colon cancer induced group attributed to the inflammatory effect of AOM that was generally associated with oxidative stress  and / or due to the neutrophil infiltration that is linearly related to the MPO activity that increased as a result of effect of DSS ingested in combination with AOM . LDH is one of the important enzymes catalyzing glycolytic pathway. Under normal conditions, it produced in the body in little amounts and increases in the tissues as a result of physiological abnormalities. Therefore, it is used as an indicator for incidence of cancers . During the current study, glucose level decreased significantly with lowering LDH activity in colon of cancer induced group. This agreed with Denko (2008) who suggested that LDH activity decreased due to increasing number of the cells that produce energy through glycolysis under anaerobic conditions by consuming great glucose amount during cancer development and therefore, the LDH consumed and hence its activity decreased. Also, Pujara and Chaudhary (2017) suggested that LDH activity decreased in the cancer tissue due to destruction of the growing cancer cells to other tissues leading to release of intracellular enzymes into blood stream and hence its activity decreased.Calcium belongs to the trace elements that change in colon cancer and their levels have prognostic significance in its incidence and progression . In the present study, calcium level increased in colon tissues of cancer induced group. This might be attributed to the oxidative stress and the disturbances of calcium homeostasis that might be related to injury of the colon tissues during the induction process . The nano-extract restored all altered measurements  assayed in colon tissues to normal values. This might refer to efficiency of the active constituents to inhibit the oxidative stress and the metabolic intermediates and hence inhibit generation of the ROS during activation of the microsomal enzyme .Malondialdehyde (MDA) is the most common end product of polyunsaturated fatty acids peroxidation that induced by oxidative stress . In the present study, the MDA level elevated significantly in colon of cancer induced group. This was attributed to ability of AOM to initiate oxidative stress through producing extremely ROS after its metabolism in the body . Furthermore, the reactive metabolites increased during the hepatic AOM metabolism and/or during the process of colon carcinogenesis well. Consequently, these metabolites stimulate production of substantial amount of H2O2 in colon tumor cells then released into the circulation . Also, the MDA induced protein damage, DNA fragmentation, gene mutations and loss of membrane integrity . Therefore, the lipid and protein oxidation products increased significantly in AOM injected group during induction of the colon cancer.M. oleifera possesses antioxidant activities due to presence of many biologically active phyto-constituents . Treatment with Ag-NPs green synthesized with M. oleifera leaves extract could protect cells from damage, demise and dysfunction that caused by the oxidative stress. This might be attributed that nano-extract is capable to protect cells by stabilizing the membrane permeability through inhibiting peroxidation reactions and preventing glutathione depletion. The antioxidant profile was restored by the nano-extract which prevented the peroxidation reactions and generation of ROS in colinic cells as presented by its ability to quench and scavenge production of the free radicals as supported by Shousha et al., (2019). In addition, our findings were supported by Dhanapal and Ping (2017) who revealed that the antioxidant capacity of M. oleifera leaves extract increased after incorporating Ag-NPs. The improved antioxidant ac¬tivity might be related to the ability of antioxidant functional groups to adsorb onto surface of Ag-NPs.During the current study, it was found that AOM caused significant decrease in TAC and activities of the antioxidant enzymes (CAT and GPx). This agreed with Burlamaqui et al., (2013) and supported by Waly et al., (2016) who suggested that AOM is able to modify activities of the antioxidant enzymes that provide the first line of cellular defense for eliminating the free radicals and play a critical role in maintaining the body’s defense mechanism against the destructive effects of ROS. Animal models were used for studying pathogenesis of colorectal tumors, as well as for evaluating efficiency of the extracts to be used as alternative treatments . In the current experiment, the colonic tissues were stained by H&E to be examined histopathologically. Throughout the colonic mucosa of the AOM-treated rats, severe histological abnormalities were noticed. This was in accordance with Sengottuvelan et al., (2006) and supported by Saleem et al., (2015) who reported that the oxidative stress that induced as a result of ROS generation with depletion of the antioxidant enzymes were responsible for inflammatory cells infiltration and the severe histopathological alterations noticed in colon cancer induced group. The alterations in architecture of colon mucosa with typical inflammatory changes in colonic architecture such as crypt and surface epithelial loss as well as infiltration of inflammatory cells and complete destruction of the epithelial architecture might be attributed to effect of DSS that administrated to rats through drinking water during the induction process . Also, these histopathological abnormalities were related to the antioxidant status of the colonic tissue that was altered as a result of excessive ROS production . Nano-extract minimized severity of the inflammatory responses that is considered as stage of colon cancer and malignancy criteria in nano-extract simult-treated group and prevented it completely in the post-treated group as compared to the other nano-extract treated ones. This might be attributed to presence of the phenolic compounds that accumulate in response to a wide range of stressors to exhibit their beneficial effect through their capability of singlet oxygen-quenching and radical scavenging activity . The nano-extract protected the colonic cells from damage and dysfunction that induced by oxidative stress due to incorporation of Ag-NPs that increased the biological efficiency and the antioxidant activity of plant extract .M. oleifera nano-extract showed beneficial effect against the electrophoretic alterations in the different native protein patterns and this might refer to presence of the active polyphenolic constituents that increased after incorporating Ag-NPs . Electrophoresis used as standard tool for separating, identifying and quantifying of different proteins based on net charge, size and shape of the protein molecules. Therefore, it is used for analyzing stoichiometry of a specific subunit of a protein complex . Each band is made up of group of the proteins that characterized by independent metabolic properties . The electrophoretic alterations may occur at qualitative level by hiding one or more of normal bands with existence of abnormal ones. Otherwise, the alterations may occur at quantitative level by altering quantities of the bands that identified at normal mobilities. The SI is only related to the physiological state and indicates the qualitative alterations and it is inversely proportional to the GD. The low SI values are associated with high GD values indicating differences in number and arrangement of the bands . During the current study, it was found that AOM caused alterations in native protein pattern. This was in agreed with Yasui and Tanaka (2009) who suggested that the native protein might be altered due to its deleterious effect on RNA transcripts, altering their amounts, localization or translation and / or due to its direct interaction of AOM with proteins. Moreover, the modifications in protein pattern might be related to altering DNA organization and hence changing DNA function and hence protein expression . Lipids are considered as one of the most sensitive cellular macromolecules . In the present study, the native lipoprotein pattern was altered severely during induction of colon cancer as a result of AOM injection. This might be attributed to accumulation the peroxidation products that attack the lipid portion through inducing formation of the ROS associated with depletion of the antioxidant defenses leading consequently to oxidative modifications and denaturation of the lipid moieties of proteins as suggested by El-Sayed et al., (2018) and supported recently by Aboulthana et al., (2020). Calcium-moiety of native protein categorized as low molecular weight acidic protein and it inhibits formation of hydroxyapatite leading to alterations in the mineralization process. In addition, it exhibits an effective role in maintaining integrity of the tissues against the toxic agents . During the present study, it was found that AOM caused alterations in calcium moieties of the native protein and this was in accordance with El-Sayed et al., (2018) who reported that the abnormalities occur in this native pattern qualitatively and quantitatively due to the oxidative stress and overproduction of the ROS that convert hydrogen atom (active) from these biologically active macromolecules. Moreover, these proteins might be altered due to the abnormal mineralization in the tissue . As obtained during the current experiment and supported recently by Aboulthana et al., (2020), the alterations in this protein pattern might be related to elevating calcium concentration in the colon tissue and this leads to stimulating the oxidative stress and hence leads to variations in this protein pattern. M. oleifera nano-extract exhibited antioxidant efficiency against the abnormalities in the electrophoretic in CAT and POX patterns. This might refer to enhancing the total polyphenolic compounds that exhibit scavenging activity against ROS attack .Antioxidant enzymes are tissue dependent and vary from tissue to tissue. Their activities might be altered due to degeneration of the protein contents a result of attack of the ROS that consequently lead to changing the metabolic pathways . In the present study, severe alterations were noticed in the electrophoretic antioxidant isoenzymes in colon of cancer induced group. This was in accordance with Aboulthana et al., (2020) who reported that ROS were responsible for changing rate of protein expression secondary to DNA damage leading to structural changes in protein portion of native enzymes. These isoenzymes remain electrophoretically integrated if there were no abnormalities in rate of the protein expression . Furthermore, the electrophoretic changes might occur due to binding of AOM to the native macromolecules inducing secondary structural changes in addition to effect of products of the oxidative stress and the peroxidation reactions . The Esterase-like albumin activity is considered as marker for many chronic diseases and expressed by several molecular forms with hydrodynamic properties and identified by their molecular weights . It can be visualized by staining the substrate (α- and β-naphthyl acetate) in the presence of dye coupler  . In the present study, alterations were noticed in the electrophoretic EST pattern and this agreed with Deabes et al., (2021b) and Aboulthana et al., (2020) who confirmed that these changes might refer to oxidative stress and generation of the ROS that targeting structure of albumin and hence lead to various mutations occurred at qualitative level by hiding one or more of normal bands with existence of abnormal ones. Scavenging of the free radicals as hydroxyl radical which is the major cause of oxidation reactions by extract may be attributed to their phenolic compounds which are the primal source of antioxidant ability . The nano-extract exhibited an important role in maintaining integrity of EST isoenzymes against the oxidative reactions due to presence of these polyphenols that stimulate the antioxidant enzymes to overcome attack of the reactive species targeting these macromolecules . TP53 gene elevated significantly in AOM induced colon cancer group due to accumulation of TP53 protein within the tumor cells. Also, elevation of the TP53 gene expression might refer to stability of TP53 protein that increased as a result of the aberrant alterations in acetylation and/or phosphorylation status of TP53 and consequently leads to suppressing it’s binding to DNA consensus sequences. Moreover, the post-translational modification is responsible for suppression of TP53 activity in colon of cancer induced . APC belongs to the most important tumor suppressor genes that expressed as a result of precancerous lesions during colon carcinogenesis and promote cellular differentiation in intestinal lineages . Elevation of the relative expression of APC gene was noticed in colon cancer induced group and this was in accordance with Grivennikov et al., (2012) who supported that level of APC gene expression was noticed greater in colon cancer induced group and this might be attributed to overexpression of the APC that is required as a central gatekeeper protein for regulating multiple pathways in colon cancer progression. Also, the changes in expression of the APC protein that is related to the alterations in expression of the APC gene lead to promoting colon cancer due to the abnormalities in cell cycle . M. oleifera nano-extract decreased the relative expression of TP53 and APC genes in the simult- and post-treated groups to normal levels. Although it could not decrease their levels in pre-treated group, it decreased their levels significantly as compared to cancer induced group. This might be attributed to efficiency of M. oleifera leaves extract to scavenge oxygen and nitrogen-based reactants generated in mitochondria, stabilizes mitochondrial membrane in addition to its ability to enhance anti-apoptotic signaling . Incorporation of Ag-NPs into plant extracts enhanced the antioxidant properties through increasing the total antioxidant capacity, total reducing power and free radical scavenging activity in comparison with the crude extracts. Moreover, the nano-extract exhibited high cytotoxicity against growth of human colon cancer cells compared to the crude ones. This might be attributed to increasing concentrations of the active phyto-constituents that have the ability to inhibit cell proliferation and induce programmed cell death for controlling growth of the malignant cells .The irregularities in cell cycle are usually responsible for cancer progression . AOM is activated in liver by Noxidation, generating reactive compounds essential for chemical carcinogenesis (metilazoximetanol ion and methyl diazoxide) being brought to the colon into the bloodstream or via bile as a glucuronide conjugate. After activation, the methylated DNA is mainly at positions N7-guanine and O6-guanine . Both of the TP53 and APC are the most sensitive genes susceptible to be mutated during incidence of colorectal cancers . During the present study, it was noticed that the relative expression ofM. oleifera leaves nano-extract ameliorated the colon cancer induced by AOM through lowering levels of the hematological and biochemical measurements and restoring their levels to normal values in all nano-extract treated groups especially in simult- and post-treated groups. It decreased levels of the tumor (CEA and CA 19-9) and inflammatory (CRP and MPO) markers to normal levels in both of nano-extract simult- and post-treated groups. Moreover, it ameliorated the antioxidant system by elevating activities of the antioxidant enzymes (CAT and GPx) with lowering products of the lipid and protein oxidation reactions (LPO and TPC) in all nano-extract treated groups and restored their levels to normalcy in nano-extract simult- and post-treated groups and hence it prevented the inflammatory reactions detected histopathologically in colon tissues and restored the tissue to its normal configuration in that groups. As regard to the electrophoretic patterns, the nano-extract exhibited improvement at qualitative level through restoring the absent normal bands with hiding the abnormal ones. At the quantitative level, it restored the relative quantities of the total bands to normal values in the nano-extract simult- and post-treated groups. Also, the molecular assay supported the findings obtained by biochemical and electrophoretic assays and emphasized that the nano-extract decreased expression of the tumor suppressor genes (TP53 and APC) and restored their expression levels to normalcy in nano-extract simult- and post-treated groups.In conclusion, Silver W.G.S. and W.M.A. proposed the research idea and designed the experimental model for this work. A.H.S. and M.H.S. were responsible for preparing the plant extract. W.M.A., E.A.E. and M.H.S. performed the experimental work and provided reagents/materials necessary for experiments. A.H.S. was responsible for preparing the silver plant nano-extract. All authors collected the theoretical details from the previous studies, interpreted, analyzed the data and draft the manuscript then they read and approved the final manuscript. W.M.A. correspond the manuscript.


Youth with existing psychiatric illness are more apt to use the internet as a coping skill. Because many “in-person” coping skills were not easily accessible during the COVID-19 pandemic, youth in outpatient mental health treatment may have been particularly vulnerable to the development of problematic internet use (PIU). The identification of a pandemic-associated worsening of PIU in this population is critical in order to guide clinical care; if these youth have become dependent upon the internet to regulate their negative emotions, PIU must be addressed as part of mental health treatment. However, many existing studies of youth digital media use in the pandemic do not include youth in psychiatric treatment or are reliant upon cross-sectional methodology and self-report measures of digital media use.This is a retrospective cohort study that used data collected from an app-based ecological momentary assessment protocol to examine potential pandemic-associated changes in digital media youth in outpatient mental health treatment. Secondary analyses assessed for differences in digital media use dependent upon personal and familial COVID-19 exposure and familial hospitalization, as well as factors associated with PIU in this population.The participants were aged 12-23 years and were receiving mental health treatment in an outpatient community hospital setting. All participants completed a 6-week daily ecological momentary assessment protocol on their personal smartphones. Questions were asked about depression , anxiety , PIU (PIU-SF-6 [Problematic Internet Use Short Form 6]), digital media use based on Apple’s daily screen time reports, and personal and familial COVID-19 exposure. The analyses compared screen time, psychiatric symptoms, and PIU between cohorts, as well as between youth with personal or familial COVID-19 exposures and those without. The analyses also assessed for demographic and psychiatric factors associated with clinically significant PIU-SF-6 scores.P=.02) and to spend more time using social media each day (P=.049). The overall amount of daily screen time did not differ between cohorts. Secondary analyses revealed a significant increase in average daily screen time among subjects who were exposed to COVID-19 (P=.01). Youth with clinically significant PIU-SF-6 scores were younger and more likely to have higher PHQ-8 (P=.003) and GAD-7 (P=.003) scores. No differences in scale scores or media use were found between subjects based on familial COVID-19 exposure or hospitalization.A total of 69 participants completed the study. The participants recruited during the pandemic were significantly more likely to meet the criteria for PIU based on their average PIU-SF-6 score (Our findings support our hypothesis that PIU may have worsened for youth in mental health treatment during the pandemic, particularly the problematic use of social media. Mental health clinicians should incorporate screening for PIU into routine clinical care in order to prevent potential familial conflict and subsequent psychiatric crises that might stem from unrecognized PIU. Significant concerns exist that youth mental health worsened during the COVID-19 pandemic. While the US emergency department visits for pediatric ailments such as asthma or otitis media decreased during the pandemic, the proportion of youth presentations related to mental health crises increased in 2020 .Researchers leading these studies have been careful to note that their study designs do not allow for the determination of causality. The pandemic has not been proven to be the direct cause of worsening psychiatric illness, despite growing evidence that pandemic restrictions likely had a significant impact on youth mental health. News outlets have featured stories from youth explicitly stating that pandemic-associated stressors such as online schooling and cancelled extracurricular activities led to a worsening of anxiety or depression . MoreoveElevated daily screen time and problematic internet use (PIU), an excessive, uncontrollable drive to continue use of the internet despite negative consequences, are both well-associated with numerous psychiatric comorbidities, including depression, anxiety, substance use, self-injurious behavior, and suicidality -9. WhileHowever, not all youth appear equally susceptible to PIU and the negative effects of screen time. Youth with existing psychiatric illness, for example, may be especially vulnerable to PIU; our prior longitudinal studies in this specific population have highlighted momentary negative correlations between cell phone engagement, PIU, and mood symptom severity, suggesting that these youth use digital media to relieve psychiatric symptoms, subsequently risking PIU development ,13. TherThis study assessed the digital media habits of 2 separate cohorts of youth (1 before and 1 during the COVID-19 pandemic) who were receiving mental health treatment in a single community health setting. Data were obtained from an existing ecological momentary assessment (EMA) smartphone protocol that collected daily information about a participant’s qualitative digital media use, PIU, and symptoms of anxiety and depression over a 6-week period. Through the examination of these collected data, our study (1) assessed how digital media use and mental health may have changed for youth in mental health treatment during the pandemic and (2) explored how personal or familial exposure to the novel coronavirus might have impacted digital media habits and mental health. Due to more limited access to nondigital coping skills during the pandemic, we hypothesized that youth in the pandemic cohort would have higher rates of PIU and spend more time on screens and social media. We also hypothesized that within the COVID-19 cohort, youth personally exposed to COVID-19 might have significantly higher amounts of daily screen and social media time due to increased awareness of the disease and subsequent avoidance of in-person pastimes.The clinical implications of this study are significant. If these high-risk youth developed a more pathological relationship with digital media during the pandemic, they may have a particularly difficult time separating from digital devices when COVID-19 restrictions are eventually rolled back in favor of in-person activities and services. Because forced separation from devices is often a trigger for parent-child conflict and can precipitate a psychiatric crisis , mental The study participants were initially recruited as part of a separate app-based EMA pilot study investigating PIU in this population  and wereData for this study had previously been collected by these authors as part of a separate app-based EMA pilot protocol that used mindLAMP for daily assessment and data collection over a period of 6 weeks. MindLAMP is a free-rein research platform that includes both an online portal system and a smartphone app . All stuThe surveys included 3 clinical scales to measure PIU (PIU-SF-6 [Problematic Internet Use Short Form 6]), depression , and anxiety . The PHQ-8 is a modified version of the PHQ-9, which omits the final question assessing suicidality due to the fact that positive responses could not be actively monitored remotely. The PIU-SF-6 is a scale validated for the measurement of PIU in youth (=.77) . WordingParticipant psychiatric diagnoses were obtained from the most recent mental health visit notes in the participant’s EHR. Youth completing the study during the COVID-19 pandemic were asked to respond to a brief survey regarding their personal and familial exposure to the novel coronavirus at the beginning and end of the study period. For the purpose of this study, family was defined as whomever the youth considered to be family, not just those individuals living with the participant.Data were downloaded from mindLAMP in the form of daily scale scores, screen and social media time, and the 3 most commonly used apps on a daily basis. For each participant, daily PIU-SF-6, PHQ-8, and GAD-7 scale scores were transformed into average scale scores. This average scale score was then used to create a secondary binary variable, which described if a participant’s average score met or exceeded standardized cut-off values when screening for clinical illness. For the PIU-SF-6, this threshold was a score of ≥15 , and ≥10Addressing the study’s first goal, logistic regressions compared the number of participants whose average scale scores met clinical cut-off values for the PHQ-8, GAD-7, and PIU-SF-6 across pre–COVID-19 and COVID-19 cohorts. While the sample size of the study is a notable limitation for this model, logistic regression models were chosen for this analysis in order to adjust for confounders of age and gender. Age is a known confounder positively associated with both youth screen time and social media use; therefore, the significant difference in age between pre–COVID-19 and COVID-19 cohorts needed to be considered in this first analysis. Due to the nonnormality of the data, Mann-Whitney tests were performed to assess for differences in mean daily screen and social media times across these groups. To correct for age differences when comparing screen and social media times across cohorts, these specific data underwent intercept adjustment for age and gender prior to conducting Mann-Whitney tests.A second set of analyses assessed for differences in screen and social media times as well as average scale scores based on COVID-19 exposure within the COVID-19 cohort alone. These analyses used the Fisher exact and Mann-Whitney tests due to the small sample size. Given the unique stress of the pandemic upon minority populations, we also assessed whether youth of color in the COVID-19 cohort had significantly different digital media use compared with nonminority youth.Finally, we sought to characterize our sample with PIU comparing participants with average PIU-SF-6 scores meeting the clinical cut-off score of ≥15 to those with scores of <15. We assessed associations between age, gender, minority status, and likelihood of having clinically significant GAD-7 and PHQ-8 scores and preexisting anxiety or depressive disorder diagnoses using the Fisher exact tests. As mentioned, Mann-Whitney tests assessed for differences in age and average daily screen or social media time. All analyses were performed using Stata   and RstuA total of 69 participants completed the 6-week study. Symptom scale data were obtained from all 69 participants, and 77% (n=53) of the participants had iPhones and provided information about their smartphone use from daily screen time reports. Demographic information is summarized in P=.02) (P=.049).Controlling for age and gender, youth in our COVID-19 cohort were more likely to meet criteria for PIU based on their PIU-SF-6 scores averaged over the 6-week study (P=.02) . The aveP=.02) ; howeverP=.01) . Overall, youth with PIU-SF-6 scores of ≥15 in our study population were significantly younger and more likely to have comorbid clinically elevated PHQ-8 and GAD-7 scores (P=.003)  . However(P=.003) .Our results demonstrate that the COVID-19 pandemic may have altered digital media habits in youth with psychiatric illness. Study participants assessed over 6 weeks during the pandemic were significantly more likely to endorse consistent feelings of problematic dependency on the internet. Because rates of preexisting anxiety and depressive disorders did not differ significantly between cohorts, the higher rates of PIU seen in the COVID-19 cohort were likely not attributable to the participants’ preexisting psychiatric disorders. However, prior studies have consistently emphasized positive correlations between PIU and active psychiatric symptoms, including in youth in mental health treatment -9,12. ThSome studies have suggested that excessive internet use and PIU directly cause adverse mental health outcomes . HoweverThe fact that average screen time did not also increase during the pandemic in our study population may reflect our participants’ high baseline rates of digital media use compared with youth without active psychiatric symptoms ,22. HoweThese findings have significant implications for the treatment of youth with psychiatric diagnoses. While it is always important for clinicians to revisit a patient’s digital media habits periodically throughout the course of treatment, the pandemic may necessitate additional screening for changes in media use. Youth struggling with their psychiatric symptoms or with a history of COVID-19 exposure may also benefit from PIU screening specifically, and their parents or guardians should be asked about conflicts arising surrounding separation from devices, particularly smartphones. A positive screen will allow for the careful development of a thoughtful, gradated media plan to help youth move back into healthier patterns of digital media use and begin intentional practice of coping skills that are independent of screens. Ideally, these youth will be more successful re-adjusting to aspects of screen-free daily living if the transition is predictable and gradual and involves youth input.Finally, social media research in this population is challenging; even in adults, the recall accuracy of daily screen time is limited , and theOur research findings cannot conclude that the pandemic was the root cause of worsening youth mental health or PIU, and the study’s small sample size is a notable limitation. Our data suggest that youth in mental health treatment were at increased risk of PIU development during the pandemic, specifically those with more severe symptoms of anxiety and depression. Moreover, those youth in mental health treatment exposed to COVID-19 endorsed greater amounts of daily smartphone use than those without a history of exposure. Based on our results, we recommend that clinicians screen high-risk pediatric patients for potential pandemic-associated changes in digital media habits as this may prevent psychiatric crises secondary to digital media-related conflict in the home or at school. From a systems standpoint, such crisis prevention measures may ease the burdens placed on our already overwhelmed psychiatric crisis teams and emergency rooms as we continue to navigate the COVID-19 pandemic.


We present a case of COVID-19–positive pediatric patient for urgent urological surgery by spinal anesthesia to avoid aerosolizing procedure.A 12-year-old, COVID-19–positive boy presented for urgent wound incision and drainage at the circumcision site. Our anesthetic plan consisted of spinal anesthesia with sedation. He was transported from the COVID-19 isolation floor to the negative pressure operating room. He was placed in lateral decubitus position and oxygen was delivered through facemask. Under sedation, spinal anesthesia was achieved at first attempt. The patient maintained spontaneous ventilation without airway intervention. Patient was recovered in the operation room then transported back to the floor.Spinal anesthesia is a safe alternative to general endotracheal anesthesia for many pediatric urology procedures. Effective team communication and preparation are keys when caring COVID-19–positive patient in perioperative setting to avoid minimize the risk to healthcare providers. COVID-19 is highly infective and can spread through contact as well as respiratory and airborne transmission. Airborne transmission via aerosolizing procedures can infect individuals more than 6 ft. away [Minimizing the risk of transmission is vital to safeguard the well-being of all Health Care Professionals (HCP) battling the COVID-19 global pandemic. The capacity for human-to-human transmission of COVID-19 continues to pose a high-risk to all HCP in the perioperative setting . When caSpinal anesthesia achieves both surgical anesthesia and analgesia without airway intervention and can be performed safely and effectively, while minimizing aerosolization . Spinal In a survey conducted by The Society for Pediatric Urology and European Society of Pediatric Urology, it was found that reluctance to accept spinal anesthesia for routine pediatric urologic procedures is multifactorial . The belThe COVID-19 pandemic has led to questions about how to best care for COVID-19 patients perioperatively . CurrentAs such, we present the case of a COVID-19–positive pediatric patient who successfully underwent urgent urological procedure with spinal anesthesia. This offers the first documented report of the use of regional anesthesia as primary anesthetic modality in a COVID-19–positive pediatric patient.A 12-year-old Latino male (BMI 25) presented 12 days post-circumcision for urgent revision surgery. The patient had no past medical history, no respiratory symptoms, and tested negative for COVID-19 at the time of his initial surgery. At his follow-up appointment, the pediatric urologist determined he required revision to avoid poor cosmetic outcome. He was subsequently brought to the hospital for incision and drainage of post-operative hematoma.On exam, the child was in clear discomfort and had a large hematoma completely surrounding the penile shaft. In adherence to Children’s Hospital Los Angeles (CHLA) guidelines, the patient underwent nasopharyngeal COVID-19 swab testing prior to proceeding to the operating room (OR). His test returned positive for severe acute respiratory syndrome coronavirus (SARS-CoV). Per institutional policy, though he was asymptomatic, the patient was presumed to be carrying the virus and be high risk for disease transmission. Shortly after his positive result, we learned that the child’s mother also had been infected with COVID-19 and was recuperating at home.After a risk-benefit discussion with the surgeon, spinal anesthesia with monitored anesthesia care (MAC) was recommended for this urgent procedure with the objective of avoiding instrumentation of the airway and minimizing the risk of airborne viral transmission. A plan was formulated together with nursing to ensure appropriate and effective communication throughout the case.Once in proper PPE, the anesthesia and nursing teams transported the patient from the COVID-19 isolation floor to the negative pressure OR. He was transported in a wheelchair, donned in a face mask and isolation gown. At the end of the procedure, the recovery room nurse and OR staff transported the patient from the negative pressure OR, back to the COVID-19 isolation floor without complication.The urgency of this case was discussed with the surgical team and it was concluded that postponing surgery because of his COVID–positive status was unacceptable due to his increased risk for long-term sequalae with his complication. A plan was made to mitigate risk to HCP using the following goals: (1) avoid airway instrumentation, (2) minimize airway manipulation, and (3) reduce overall risk of viral airborne transmission. As a successful spinal anesthetic does not require airway instrumentation or general anesthesia, it was especially suited to this urgent procedure . The pla2 monitoring. Sedation for spinal placement was achieved with propofol infusion at 35 mcg/kg/min and incremental doses of ketamine (24 mg) and dexmedetomidine (16mcg).The patient’s back was prepped and draped in the usual sterile fashion at the level of the L3/L4 interspace. Lidocaine 1% (2 mL) was used to provide local anesthesia to the skin. A Gertie-MarxTM 25-gauge spinal needle was used to access the spinal canal with return of clear cerebrospinal fluid (CSF).Premedication was not provided until arrival in the OR to avoid potential sedation and respiratory depression that could necessitate airway intervention during transport. Standard anesthesia monitors were placed prior to administration of premedication. Spontaneous ventilation was confirmed with capnography and end-tidal COSpinal anesthesia was achieved on the first attempt within 5 min. A total of 2 mL, consisting of bupivacaine 0.75% with dextrose 5% (1.5 mL) and fentanyl PF 25 mcg (0.5 mL) was given. The pediatric urologist then proceeded with their portion of the procedure. Surgical anesthesia was tested prior to incision and no response to noxious stimuli was appreciated. Surgery proceeded for 45 min with the patient breathing spontaneously throughout. There were no anesthetic complications or need for airway manipulation. The child was able to move his feet prior to discharge from the OR.Adequate preparation and communication proved essential in caring for this patient with COVID-19 . CarefulPost-operation discussion with the parent and patient highlighted the importance of patient-doctor rapport. Both the parent and patient were aware of the perioperative plan which they said served as a form of anxiolysis and facilitated the child’s cooperation. The family and patient were satisfied with the care provided and appreciated the Anesthesiologist’s effort to evaluate and minimize the potential risk of this urgent case.This is the first documented use of spinal anesthesia as primary anesthetic modality in a pediatric patient with novel COVID-19. Spinal anesthesia achieves both surgical anesthesia and analgesia without airway intervention . This reAt this time, there is no consensus on anesthetic management of patients with COVID-19. A literature review of 690 articles in PubMed, using the keywords “COVID-19 pediatric surgery” left much to be desired. Five articles discussed the clinical findings associated with COVID-19 infection in children and one article indicated that multisystem inflammatory syndrome, which is common in children with COVID-19, may have cardiac involvement , 14.Advances in the realm of pediatric regional anesthesia continue to evolve with excellent safety profiles described in society guidelines . New datSpinal anesthesia is a safe alternative to general endotracheal anesthesia for many pediatric urology procedures . Data frWhile the management of pediatric patients with COVID-19 must be individualized, spinal anesthesia should be strongly considered as an alternative to general anesthesia for lower abdominal and lower extremity surgery in COVID-19–positive pediatric patients.


Scanning electron microscopy (SEM) has contributed to elucidating the ultrastructure of bio-specimens in three dimensions. SEM imagery detects several kinds of signals, of which secondary electrons (SEs) and backscattered electrons (BSEs) are the main electrons used in biological and biomedical research. SE and BSE signals provide a three-dimensional (3D) surface topography and information on the composition of specimens, respectively. Among the various sample preparation techniques for SE-mode SEM, the osmium maceration method is the only approach for examining the subcellular structure that does not require any reconstruction processes. The 3D ultrastructure of organelles, such as the Golgi apparatus, mitochondria, and endoplasmic reticulum has been uncovered using high-resolution SEM of osmium-macerated tissues. Recent instrumental advances in scanning electron microscopes have broadened the applications of SEM for examining bio-specimens and enabled imaging of resin-embedded tissue blocks and sections using BSE-mode SEM under low-accelerating voltages; such techniques are fundamental to the 3D-SEM methods that are now known as focused ion-beam SEM, serial block-face SEM, and array tomography . This technical breakthrough has allowed us to establish an innovative BSE imaging technique called section-face imaging to acquire ultrathin information from resin-embedded tissue sections. In contrast, serial section SEM is a modern 3D imaging technique for creating 3D surface rendering models of cells and organelles from tomographic BSE images of consecutive ultrathin sections embedded in resin. In this article, we introduce our related SEM techniques that use SE and BSE signals, such as the osmium maceration method, semithin section SEM (section-face imaging of resin-embedded semithin sections), section-face imaging for correlative light and SEM, and serial section SEM, to summarize their applications to neural structure and discuss the future possibilities and directions for these methods. Scanning electron microscopy (SEM) enables images to be obtained by detecting various signals  that escape from specimens when an incident electron probe emitted from an electron gun strikes the observation targets. Among these signals, both SE and BSE signals are most commonly used in biological and biomedical research. SEs are emitted near the surface of specimens and provide surface information on tissues and cells. SEs that are produced in deeper regions of the samples are absorbed because of their low energy. More SEs escape from projections, such as microvilli than from the flat surface of specimens. This phenomenon is called the edge effect, which contributes to the image formation of the three-dimensional (3D) surface topography of specimens; the fine protrusion of the cytoplasm appears bright on the image, whereas flat areas appear dark. Various SEM techniques that use SE signals for bulk specimens have been developed by numerous SEM researchers, which include the conventional technique as well as hydrochloric acid digestion , imaging of both resin-embedded tissue blocks (block-face imaging) and resin-embedded sections (section-face imaging) is possible with SEM using a BSE detector (BSE-mode SEM). These novel BSE imaging techniques have broadened the biological applications of SEM and have been applied to recent 3D-SEM techniques, such as serial block-face SEM . Section-face imaging of resin-embedded semithin sections  using BSE-mode SEM provides high-resolution ultrathin images that are comparable to conventional transmission electron microscopy (TEM) images of ultrathin sections without requiring ultramicrotomy  were used in accordance with the Guidelines for the Care and Use of Laboratory Animals  under the permission of the experimental animal welfare committee of Asahikawa Medical University.4 (0.1 M PB). Specimens were then cryoprotected in 50% dimethyl sulfoxide (DMSO), frozen on an aluminum plate that was precooled with liquid nitrogen, and cracked into two pieces using a screwdriver and hammer. Several pieces of tissues were subsequently thawed in the DMSO solution. To remove soluble cytoplasmic proteins from the cracked surface of cells, the tissues were immersed in 0.1% OsO4 (0.1 M PB) at 20°C for several days. The macerated tissues were then processed for conductive staining; the specimens were immersed in 1% OsO4 solution, treated with 1% tannic acid, and further soaked in 1% OsO4. After conductive staining, they were dehydrated in a series of graded ethanol, transferred to isoamyl acetate, and dried in a critical point dryer . The dried specimens were mounted onto aluminum bases, coated with platinum-palladium in an ion-sputter coater , and observed through a scanning electron microscope . Scanning electron microscopes that are equipped with field emission (FE) guns provide sufficient information on the 3D ultrastructure of macerated tissues. Details of the protocol for the osmium maceration method are available in previous publications  using a diamond knife . Subsequently, these sections were picked up using aluminum loops  and mounted on glass microscope slides. Semithin sections were adhered to the glass slides by heating on a hot plate (60°C) and stained with toluidine blue, and the regions of interest (ROI) of tissues were photographed using light microscopy (LM). Following LM observation, sections were stained with heavy metals , and the sections on the glass slides were mounted onto aluminum bases. The sections were then coated with carbon using a carbon coater , and the same regions of the specimens previously photographed using LM were acquired using BSE-mode SEM under an accelerating voltage of 2–2.5 kV . The black and white reversed images of the resin-embedded semithin sections captured by BSE-mode SEM resemble TEM images of ultrathin sections. Detailed protocols for semithin section SEM are provided in our original article , and coated with carbon for conductive treatment. The ROI of the serial sections was then imaged using BSE-mode SEM . For the 3D reconstruction of the structures of interest, serial tomographic images acquired using SEM were loaded into the Amira software  and aligned using the align tool. The aligned images were subsequently imported into the Photoshop software , and the structures of interest  were segmented by delineating their boundary contours. The segmented images were then reimported into Amira, and 3D surface rendering images were created. Detailed information on serial section SEM and the 3D reconstruction method is available in our original articles  type,” and the “in-lens-type.” Scanning electron microscopes equipped with a snorkel lens are most suitable for the seamless analysis of resin-embedded sections from low to high magnification.The conductive treatment of metal coating is critical to the preclusion of charging artifacts during SEM observations of resin-embedded sections mounted on glass slides. Platinum and platinum-palladium, which are targets for metal coating, are usually used for SE-mode SEM of balk specimens, but they are unsuitable for high magnification observations because their particles are visible using BSE-mode SEM under >50,000-fold  or fixed by perfusion with an aldehyde fixative and immersion with OsO4 , are removed by soaking tissues in diluted OsO4 for several days. This preparation step is termed the osmium maceration procedure, which is a pivotal process in selectively maintaining the membranous organelles in the cells. Usually, a high-concentration solution (1%–2%) of OsO4 is used as the fixative for cell membranes. It is well established that OsO4 reacts with lipid molecules of membrane components. However, it is less known that diluted OsO4 also has the potential for removing cytoplasmic soluble proteins from the cytoplasm.Conventional preparation methods for bulk specimens using SE-mode SEM have been applied to analyze the 3D surface topography of tissues and cells. To visualize the intracellular structure using SE-mode, SEM researchers have introduced various cracking techniques, such as ethanol and DMSO freeze fracturing  at 20°C in accordance with the original protocol of the osmium maceration method developed by Tanaka and colleagues , which requires a high level of skill that is acquired with long-term training. To overcome these technical problems of TEM, we recently established a novel imaging technique termed semithin section SEM  for acquiring ultrathin images that are comparable to those acquired using conventional TEM by observing resin-embedded semithin sections using BSE-mode SEM. It requires no expert techniques, such as ultramicrotomy  have allowed us to obtain information on resin-embedded ultrathin sections using BSE-mode SEM  white resin is an alternative embedding medium to epoxy resin and is widely used for post-embedding immunohistochemistry and immuno-EM , which combines two different probes, such as nanogold and fluorescence dyes , allow us to obtain more detailed information on the localization of target molecules imaged using LM at the EM level  as the embedding material, which offers further opportunities for using immunocytochemical approaches with SEM , we cannot neglect other valuable techniques for SE-mode SEM  that provide useful information on the 3D structure of tissues and cells. Accurate comprehension of the ultrastructure of bio-specimens requires comparisons of tissue micrographs prepared using various morphological approaches. In the future, we aim to establish novel SEM methods by combining various morphology or biochemical techniques with our EM techniques, which will help clarify the 3D ultrastructure of neural structures as well as organelles.DK and SK performed the experiments and wrote the original draft. TW reviewed and revised the manuscript. MS reviewed the manuscript. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.