The glucose uptake in skeletal muscle is essential to produce energy through ATP, which is needed by this organ to maintain vital functions. The impairment of glucose uptake compromises the metabolism and function of skeletal muscle and other organs and is a feature of diabetes, obesity, and ageing. There is a need for research to uncover the mechanisms involved in the impairment of glucose uptake in skeletal muscle. In this study, we adapted, developed, optimised, and validated a methodology based on the fluorescence glucose analogue 6-NBDG, combined with a quantitative fluorescence microscopy image analysis, to determine the glucose uptake in two models of skeletal muscle cells: C2C12 myotubes and single fibres isolated from muscle. It was proposed that reactive oxygen and nitrogen species (RONS) and redox homeostasis play an important role in the modulation of intracellular redox signalling pathways associated with glucose uptake. In this study, we prove that the prooxidative intracellular redox environment under oxidative eustress produced by RONS such as hydrogen peroxide and nitric oxide improves glucose uptake in skeletal muscle cells. However, when oxidation is excessive, oxidative distress occurs, and cellular viability is compromised, although there might be an increase in the glucose uptake. Based on the results of this study, the determination of 6-NBDG/glucose uptake in myotubes and skeletal muscle cells is feasible, validated, and will contribute to improve future research. Skeletal muscle is the largest organ in the body and exerts different functions such as locomotion, equilibrium, heat generation, and metabolism, which involve a continuous demand for and consumption of energy ,2. ATP iSkeletal muscle fibres are the main type of cells that constitute skeletal muscle and are postmitotic multinuclear cells formed from the fusion of single cells, myoblasts, which differentiate into myotubes and finally mature to become fibres ,8. As me3H] glucose [Glucose uptake in skeletal muscle has been studied using different experimental models in vivo, in vitro, and ex vivo, focusing on biological samples such as whole muscle, muscle biopsies, and cellular models. Furthermore, the analytical determination of glucose uptake has been carried out using different analytical techniques. The most frequent include biochemical techniques, such as those associated with the enzymatic activity of glucose oxidase , radiois glucose ,15, nucl glucose , and flu glucose ,15,16,17In this study, we implement suitable and feasible methodological approaches that will provide a robust tool to determine glucose uptake in skeletal muscle. We determine the glucose uptake in two models of skeletal muscle. One is the C2C12 myotube model, whose cells are obtained from the fusion (differentiation) of C2C12 myoblasts , and the other is a model of single mature skeletal muscle fibres isolated from the mouse flexor digitorum brevis muscle. The analytical technique used to determine the glucose uptake in our models is based in the use of a fluorescence glucose analogue, 6-NBDG amino)-6-Deoxyglucose). 6-NBDG is a fluorescence nonhydrolyzable glucose analogue that is transported across the plasma membrane by the glucose transporter GLUT4 in skeletal muscle and retained in the cell without any transformation or metabolisation ,19. ThisThe main aim of the study is the development, optimisation, and validation of the methodological approaches to determine glucose uptake in the model of C2C12 myotubes since it is more feasible and avoids the use of animals. The second part of the study involves the application and adaptation of this methodology to a model of single skeletal muscle fibres isolated from flexor digitorum brevis (FDB) mouse muscle, which is more appropriate to study glucose uptake in pathological conditions associated with insulin resistance that appear in pathologies such as diabetes, obesity, and ageing. In parallel, we will investigate how RONS and redox homeostasis might affect the glucose uptake in skeletal muscle. As mentioned above, we hypothesised that RONS plays an important role as mediators in glucose uptake. The current knowledge in this area is scarce and limited. However, it is promising in the field of redox biology that the activity of RONS and redox homeostasis might be potential targets for the therapeutic intervention of insulin resistance in diabetes, obesity, and ageing ,12.2O2 presented values of fluorescence that were slightly lower than those of the positive control myotubes, and the result was significant in the case of the 10 μM H2O2 group. When myotubes were concomitantly treated with bovine insulin and exposed to H2O2, fluorescence increased slightly compared to corresponding untreated myotubes. We observed that 6-NBDG provided fluorescence to myotubes, indicating that this glucose, 6-NBDG, was incorporated into the myotubes, and bovine insulin led to a slight increase in glucose uptake. Conversely, H2O2 might decreased glucose uptake under these experimental conditions  that was significantly lower compared from that of myotubes incubated with 6-NBDG (positive control). Myotubes treated with bovine insulin showed higher fluorescence compared to untreated myotubes (positive control). Myotubes exposed to Hnditions .2O2 for 2 h showed higher values of fluorescence that were significant in myotubes exposed to 10 μM H2O2. Myotubes treated with insulin and exposed to H2O2 concomitantly showed lower fluorescence compared from the myotubes treated with insulin alone, with significant results observed for myotubes treated with insulin and exposed to either 10 or 100 μM H2O2. Therefore, there was confirmation that human recombinant insulin produces glucose uptake in the model of myotubes and that 2 h of exposure to hydrogen peroxide also improves glucose uptake. However, there might be an interaction between insulin and hydrogen peroxide, since the expected increase in glucose uptake induced by insulin did not appear when myotubes were exposed concomitantly to H2O2 , a glutathione oxidant, produced an increase of fluorescence compared from control myotubes, which was similar to the fluorescence of myotubes pre-treated with a glutathione donor, GSHEE, and diethyl maleate. Moreover, when myotubes were pre-treated with insulin, instead of GSHEE, the fluorescence appeared to be higher. Myotubes treated with diamide, which depletes intracellular GSH, showed a dramatical increase in fluorescence compared from the control myotubes. However, when myotubes were pre-treated with the glutathione donor GSHEE and diamide, fluorescence decreased, although the levels were still higher compared to those of the control. The pre-treatment of myotubes with insulin instead of GSHEE produced higher fluorescence in myotubes. The depletion of glutathione, the main intracellular antioxidant, appears to facilitate a prooxidant intracellular environment that favoured glucose uptake, which might be additive to the glucose uptake induced by insulin. When the oxidant environment became attenuated, at least partially due to the replenishment of GSH by GSHEE, the increase in glucose uptake was partially reduced. In addition, we observed that either DEM or diamide produced toxicity in myotubes, which was avoided when myotubes were pre-incubated with glutathione donor GSHEE. Insulin appeared to protect against toxicity induced by DEM but not against toxicity induced by diamide .The treatment of myotubes with cytochalasin B, which is an inhibitor of glucose transport, did not produce changes in fluorescence. However, when myotubes were concomitantly treated with insulin and cytochalasin B, fluorescence of the myotubes decreased, although this decrease was not significant. Myotubes treated with diethyl maleate showed higher fluorescence compared to the control nontreated myotubes, although not statistically significant. The concomitant treatment with diethyl maleate and cytochalasin B produced a significant increase in fluorescence. The treatment with diamide produced a dramatically significant increase in fluorescence, and this effect remained when myotubes were cotreated with diamide and cytochalasin B. It appears that cytochalasin B might decrease the glucose uptake produced by insulin. In contrast, cytochalasin B might potentiate the glucose uptake induced by diethyl maleate and was not observed to affect the increase in glucose uptake produced by diamide .p > 0.05), the fluorescence of fibres treated with insulin was higher than that of the control. Moreover, the fluorescence of fibres exposed to hydrogen peroxide and that of fibres treated with angiotensin II were higher than those of the control. Insulin, hydrogen peroxide, and angiotensin II appeared to favour glucose uptake in these fibres.p > 0.05), the fluorescence of fibres treated with insulin is similar here to the fluorescence of the control fibres. However, the fluorescence of fibres exposed to hydrogen peroxide was higher than the fluorescence of the control fibres, considering there is not statistical difference. In the case of fibres treated with SNP, there was only one viable fibre, which presented an atypical morphology. Although it might be unreliable accounting just one fibre for any comparison, the fluorescence of this fibre was lower compared to the control group. Insulin was not found to facilitate glucose uptake in these fibres. However, hydrogen peroxide might slightly favour glucose uptake, assuming all statistical reservations.The main aim of this study was to develop a feasible methodology to determine glucose uptake in live skeletal muscle cells. This methodology is based on three pillars: (i) a glucose analogue, 6-NBDG, which is resistant to being metabolised and emits fluorescence; (ii) two models of live skeletal muscle cells, C2C12 myotubes and isolated mature fibres; and (iii) a quantitative fluorescence microscopy imaging analysis system used to determine 6-NBDG/glucose uptake by cells.In the first instance, we assessed whether this methodology might detect glucose uptake in the model of C2C12 myotubes. These skeletal muscle cells were treated with insulin, the main physiological stimulus for glucose uptake. In addition, other myotube cultures were exposed to different concentrations of hydrogen peroxide. Several recent studies indicated that ROS may modulate and favour glucose uptake ,10,11, a2O2 at different concentrations in the presence of 6-NBDG for at least 19 h (overnight). In this experimental set  for 2 h and proved that 6-NBDG uptake increased compared to the control myotubes . Howeverntal set , we obsental set , where 6lisation ,19 and p2O2: 1, 10, and 100 μM  growing in the culture was close to 100% and, in practise, it is very difficult to find areas without cells in microscopy field images that could be selected to quantify the fluorescence of the medium. In the case of fibres, it is always possible to find an extracellular area close to the fibre that is free of cells and could be selected to quantify the fluorescence of the medium (background).2) that affect glucose uptake. The fibres treated with the ROS hydrogen peroxide yielded a significant increase in 6-NBDG, which confirmed that hydrogen peroxide and a moderate oxidative state facilitated glucose uptake in skeletal muscle fibres, although these fibres might be insulin resistant. This result reinforces the hypothesis that oxidative eustress produced by hydrogen peroxide [The first set of experiments was performed with fibres isolated from 9–10-month-old mice . Fibres peroxide  facilitaperoxide . This isWe performed a set of experiments with fibres isolated from 19-month-old mice . Fibres The last set of experiments examined skeletal muscle fibres isolated from very old mice, 25–29 months old. It was observed that 6-NBDG uptake in fibres treated with insulin was similar to that in the control fibres without treatment, whereas hydrogen peroxide appeared to slightly improve 6-NBDG uptake. In the case of the nitric oxide donor SNP, there was only one fibre that showed an atypical morphology while other remaining fibres appeared damaged, with signs of toxicity or damage. This might be due to the increase in the intracellular concentration of nitric oxide in these fibres making such fibres prone to the reaction of nitric oxide with superoxide to generate peroxynitrite, which is a very reactive RONS that produces deleterious oxidative damage in proteins and lipids and might produce the toxicity and damage observed in fibres . It appeTM, Invitrogen, Thermo Fisher Scientific .The hydrogen peroxide solution (30 wt% in water), S-nitroso-N-acetylpenicillamine (SNAP), Sodium nitroprusside dihydrate (SNP), NOC-7, Diamide, Diethyl Maleate (DEM), Glutathione reduced ethyl ester (GSHEE), Superoxide dismutase (SOD), Angiotensin II, Cytochalasin B, bovine insulin, and recombinant human insulin were obtained from Merck-Sigma-Aldrich . Insulin receptor antagonist peptide (S961) was obtained from Phoenix Pharmaceuticals, Inc. . 6- amino)-6-Deoxyglucose (6-NBDG) was obtained from Molecular Probesv/v) foetal bovine serum (FBS) , 2 mM L-glutamine (Sigma-Aldrich), 50 i.u. penicillin, and 50 µg of mL-1 streptomycin (Sigma-Aldrich) at 37 °C in an atmosphere of 5% CO2 under adequate humidity. C2C12 myoblasts were differentiated for 7 days to obtain myotubules; during this time, the myoblasts fused and transformed into multinuclear myotubes with a tubular morphology. Differentiation involved growing myoblasts to 80%–90% confluency and then replacing the growth medium with a differentiation medium (DMEM with 2% (v/v) horse serum (Invitrogen) supplemented with 2 mM L-glutamine, 50 i.u. penicillin, and 50 µg mL-1 of streptomycin) [The C2C12 mouse skeletal myoblasts were purchased from the American Type Culture Collection . C2C12 was cultured in Dulbecco’s modified Eagle’s medium  containing 10% (tomycin) .Female and male 9–29-month-old C57BL/6J mice were used in this study. The procedures involving animals were approved by the Bioethics Committee of the University of Salamanca and conducted in accordance with the Spanish (RD 53/2013) and European Union (2010/63/EU) guidelines for animal experimentation.g for 30 s) four times. After each centrifugation, the supernatant was removed and replaced with fresh culture medium. Washed fibres were seeded onto a 13 mm diameter glass surface inserted in individual 35 mm culture dishes . The glass surface was previously coated with a collagen matrix . Finally, fibres were incubated for 18–24 h covered with culture medium at 37 °C in 5% CO2 in a humidified atmosphere to allow adherence of the fibres to the collagen matrix. Fluorescence microscopy experiments were undertaken only on selected fibres that displayed excellent morphology and exhibited clear striations along the sarcolemma.Single skeletal muscle fibres were isolated from the FDB muscle of the mice according to the protocol established by Palomero et al. ,54,56,612-2H2O, 1.5 mM MgSO4-7H2O, 1.25 mM NaH2PO4, 26.2 mM NaHCO3, and 11.1 mM glucose; pH 7.4). After one hour of stabilisation, we added the different reagents for the experimental treatments: bovine and recombinant human insulin, SNAP, SNP, Diamide, GSHEE, SOD, Angiotensin II, NOC-7, S961, DEM, Cytochalasin B, and hydrogen peroxide. After one hour, 6-NBDG was added to the cell medium and incubated with the reagents of treatments for another hour. After this time, in some experiments . In the case of skeletal muscle fibres, the complete procedure was undertaken with fibres incubated overnight in Minimum Essential Medium Eagle  with 10% (v/v) foetal bovine serum (Invitrogen) supplemented with 2 mM L-glutamine, 50 i.u. penicillin, and 50 µg mL−1 of streptomycin (Sigma-Aldrich)). After overnight incubation with the reagents for treatments and 6-NBDG, both myotubes and fibres in the culture plates were placed under the fluorescence microscope stage for the registration of images.C2C12 myotubes and skeletal muscle fibres were exposed to a similar procedure. First, the C2C12 cell medium was changed to a Krebs solution  that generated 470 nm monochromatic light. Fluorescence images were obtained through a fluorescence cube with a 450/40 nm excitation filter, 495 nm beam splitter, and 525/50 nm emission filter. Fluorescence images were acquired with a computer-controlled CCD camera  coupled to the microscope. The fluorescence images were acquired with the minimum exposure time that provided images with enough quality for quantification to avoid light-induced damage to the cells. Time-lapse images were acquired with the same exposure time, which was maintained for each time-lapse experiment performed under the same conditions. The fluorescence image analysis was undertaken using the software  included with the microscopy equipment. This software provided the fluorescence quantification of myotubes and fibres from fluorescence images that had been recorded during the time-lapse experiment. For quantification, a region of interest (ROI) from a myotube or fibre that emitted fluorescence was selected. The software provided the measurement, in terms of grey activated pixels, of the fluorescence intensity from that ROI in every image recorded. The fluorescence intensity from a unique ROI was calculated as the mean of the 6 sequential images registered from the lime-lapse of 5 min, and this was considered as the fluorescence of that particular myotube or fibre. In the case of experiments with myotubes, one time lapse was performed per myotube culture dish, and a maximum of 5 individual myotubes in the image field were selected for quantification. In the case of fibres, one time-lapse per fibre culture dish was performed, and 1–3 viable fibres in the image field were selected for fluorescence quantification. Moreover, a ROI from the extracellular space of fibres was selected to measure fluorescence, and this fluorescence (background) was subtracted from the fluorescence of the fibre to obtain the net fluorescence of the fibre, which was then used for the subsequent analysis.C2C12 myotubes and single skeletal muscle fibres were placed under a fluorescence microscope  equipped with a chamber to maintain the temperature at 37 °C in a 5% COIn addition, the light transmission images registered were used for the assessment of toxicity and viability, which might have been affected by the experimental interventions, in either myotubes or fibres. This assessment was based in the observation of morphology, density, and general aspect of myotubes and fibres.t-test) to detect differences between the two experimental treatments. The statistical difference was set at p < 0.05. The IBM SPSS Statistics software, version 28.0.1.1 (14), was used for statistical analysis.The values for the raw fluorescence of myotubes and net fluorescence of fibres, are presented as the mean ± standard error of the mean (SEM) and the number (n) of myotubes or fibres used for statistical analysis. Data were analysed with a one-way ANOVA to detect differences between experimental treatment groups, followed by a post hoc LSD test  that might interfere with normal metabolism. In addition, glucose uptake was determined over a long period (20 h), which helped explore the effects of all treatments on the improvement of glucose uptake.The method of 6-NBDG was suitable to determine an increase in glucose uptake greater than the basal glucose uptake. However, this methodology overestimated glucose uptake when basal glucose uptake was impaired .The prooxidative intracellular redox environment produced by RONS such as hydrogen peroxide and nitric oxide, which might be associated with oxidative eustress, improved glucose uptake in myotubes and skeletal muscle fibres. However, when oxidation was excessive , cells could become damaged or experience toxicity, compromising cellular viability. Moreover, in this circumstance, glucose uptake appeared to be elevated.In this article, we presented a new methodologic approach to determine 6-NBDG/glucose uptake in skeletal muscle cells. This methodology is feasible and valid and could be complemented with other research techniques, e.g., molecular biology and immunocytochemistry, to help uncover the mechanisms that produce a pathological state of skeletal muscle, insulin resistance, and impairment of glucose uptake, which are manifested in pathologies such as type 2 diabetes, metabolic syndrome, and obesity, as well as in ageing.


Depression is a serious mental health issue and the largest contributor to disability worldwide. Elderly people with depression are significantly more likely to experience negative outcomes such as poor physical health, strained social relationships, and decreased quality of life. Studies on geriatric depression are limited in developing nations like Ethiopia.The purpose of this study was to determine the prevalence of depressive symptoms and associated factors among older adults in Yirgalem, Southern Ethiopia, in 2022.P-value less than 0.05.A community-based cross-sectional study was conducted on a sample of 628 older adults in Yirgalem town from May 15 to June 15, 2022. The study subjects were selected using a multi-stage systematic sampling technique. Data were collected using the 15-item Geriatric depression scale through face-to-face interviews. The collected data were edited, cleaned, coded, and entered into Epi data version 4.6 software and analyzed using STATA version 14. Bivariable and multivariate logistic regression analysis was computed to identify factors associated with depression, and statistical significance was declared at a 95% confidence interval with a P-value of less than 0.05.A total of 620 older adults were included in the study, with a response rate of 97.8%. The prevalence of depressive symptoms among older adults was 51.77% (95% CI: 47.83–55.69). Being a woman ; being of more advanced age: 70–79 years old , 80–89 years old , 90 and older ; living alone ; having a chronic illness ; having anxiety ; and having poor social support  were statistically associated with depressive symptoms at a This study found that depression affects more than half of the elderly residents in the study area. More advanced age, being a woman, living alone, having a chronic illness, having anxiety, and having poor social support were all strongly linked to depression. There is a need to integrate counseling and psychiatric services into the community healthcare system. Aging is a universal, progressive, and detrimental process that occurs throughout one’s life. It is a naturally occurring phenomenon, and getting older is the stage in life when most aspects of life require the assistance of others . High raDepression is manifested primarily by a depressed mood or a lack of interest in previously pleasurable activities. Furthermore, depressed people may have reduced energy; difficulties with concentration, sleep, appetite, and weight changes; feelings of regret and dejection; and suicidal ideation . DepressGlobally, depression is a serious mental health issue, the third leading cause of disease burden, and the largest contributor to disability . As per A review of 42 studies revealed that the prevalence rate of depressive disorders among the elderly population is approximately 31.7% . A meta-There are several risk factors affecting depression in older age groups, which vary contextually. The risk of having depression is higher among the elderly who are women; are of advanced age; never married; are widowed, divorced, or separated; have a low education status; and are unemployed and low-income –18. StudThe complications of depression are more severe among elderly people than among their middle-aged counterparts. Depression significantly increases the risk of negative outcomes in older adults, such as cognitive deficits, disrupted social functioning, and disability, resulting in a greater burden on family and caregivers , 25.Elderly people suffering with depression have reduced life satisfaction and quality, social deprivation, increased utilization of health services and associated costs, and limitations to activities of daily living (ADL). Furthermore, it can negatively affect the prognosis and recovery of other chronic illnesses by amplifying the perception of poor health and interfering with their adherence to medications .Consequently, depression in older people significantly increases mortality related to suicidal and non-suicidal causes compared to their younger counterparts , 28. AltThe research was carried out in Yirgalem town, which is one of the oldest settlements in Sidama National Regional State, Southern Ethiopia. Geographically, the town is situated approximately 325 kilometers from Addis Ababa, the nation’s capital, and 47 km from Hawassa City, the state seat of Sidama National Regional State. According to the 2019 Yirgalem town health office report, the entire population of the town is predicted to be 79,605, with 39,166 men and 40,439 women. The data from local health extension workers showed that there were 3,236 older adults in the town. The town contains 13 kebeles , one governmental general hospital with a psychiatric clinic, and one health center. The study was conducted from May 15 to June 15, 2022.A community-based cross-sectional study design was employed. All elderly people (aged 60 and above) who resided in Yirgalem town, Southern Ethiopia were considered the source population. The study participants were elderly people who had been living in Yirgalem town, Southern Ethiopia during the 6 months before the data collection period and who were available during the data collection period. Older adults who were unable to communicate and seriously ill when the data were collected were excluded from the study.When determining the sample size for this study, the outcome variable and the variables that were significantly associated with the outcome variable were taken into consideration. The sample sizes for the first and second objectives were calculated separately. A single population proportion was employed to calculate the sample size for the first specific objective of the study. In this study, we used the proportion of depression from the study conducted in North Shoa, which was 54.6% .Therefore,Since we used a multistage sampling technique by taking a design effect of 1.5, and adding a 10% non-response rate, the calculated final sample size was 628.p = 0.05). Therefore, this study used the single population formula of the sample size since 628 is greater than the sample size calculated by the double population formula.The sample size for the second specific objective was determined using the Epi Info version 7, considering the factors that were significantly associated with depression at  was determined by the study population  divided by sample size (1459/628) = 2.3). Then, the data were collected from every two older adults. The lottery method (simple random sampling technique) was used to select the first elderly participant, and one eligible individual was selected from each household using the lottery method for the case where more than one older adult was living in the house.The Data were collected using a structured questionnaire in Amharic. The questionnaire has five sections: the first contains socio-demographic characteristics; the second contains the dependent variable depression and was measured by the Geriatric Depression Scale (GDS-15) in the form of dichotomous variable (yes/no).The geriatric depression scale has 15 items, five of which are negatively worded questions. Even though GDS is not validated in this country, several studies from Asian and many African countries with diverse sociocultural backgrounds were employed to screen depression. It has been tested and validated among older Asian adults, with sensitivity of 97%, specificity of 95%, and a Cronbach alpha value of 0.80 . In thisAnxiety was assessed using the GAD-7 tool, a seven-item questionnaire to measure the level of anxiety in the respondents during the preceding 2 weeks. Each item is scored from 0  to 4 (nearly every day) and scores of all items are added to get the total scores, ranging from 0 to 21, with a cut point of 10 and above . The toovia face-to-face interviews, using the Amharic version of the structured questionnaire, by six BSc nurses. Data collectors and the supervisor were trained on the purpose of the study, consent forms, maintaining confidentiality, and the overall data collection procedures.Stressful life events were assessed using the List of life-threatening experiences (LTE). The LTE includes 12 significant life events that occurred over the previous 6 months, which are rated by yes or no responses, with a cut point of one. The LTE has high test-retest reliability (Kappa: 0.61–0.87) as well as predictive validity, and numerous Ethiopian studies have employed LTE to measure adverse life events in various populations . The lasThe questionnaire was initially prepared in English and translated into the Amharic language for the participants by two language experts. The pre-test was conducted on 5% of the study sample in Wendo Genet town 1 week before the data collection. Training was delivered to data collectors and supervisors by the principal investigator about how to use questionnaires, the ethical principle of confidentiality, and data management prior to data collection. The data collectors were supervised daily, and the filled questionnaires were checked daily by the supervisors and the principal investigator for completeness.Data were checked, coded, and entered into Epi-Data version 4.6 and exported to Stata version 14 for analysis. The socio-demographic characteristics and other factors of respondents were analyzed by descriptive statistics.p-value of less than 0.2 at bivariable logistic regression analysis were entered into the multivariable logistic regression model. A p-value of <0.05 was considered statistically significant, and the adjusted odds ratio (AOR), with a 95% confidence interval (CI), was calculated. The goodness of fit model was checked using the Hosmer-lemshow test.Bi-variable logistic regression analysis was performed to identify the association of each independent variable with the outcome variable. All variables with a Depression: A score of five and above on the Geriatric Depression Scale GDS-15) ( (5).Anxiety: Participants who scored 10 and above on the Generalized Anxiety Disorder Scale (GAD-7) were considered as having anxiety.Social support: The Oslo-3 social support scale was used to score social support on a scale ranging from 3 to 14 with three categories: scores of 3–8 were considered poor support, 9–11 moderate support, and 12–14 strong support .Stressful life event: Using the List of Threatening Experiences (LTE), if the respondent had experienced one or more major life events within the previous 6 months from a total of twelve items, they were considered as having experienced a serious life event .Previous use of substances: Having used at least one specific substance for a non-medical purpose at least once in a lifetime  .Current Substance use: Having used at least one specific substance for non-medical purposes within the last 3 months  .From a total of 628 samples, 620 participants were included in the study, with a response rate of 98.7%. There were more men, at 52.25 % (324). The mean age and standard deviation of the study participants were found to be 72.8 and ±9.83 years, respectively, with ages ranging from 60 to 96 years. Regarding the living arrangements of participants, 83.71% (519) were living with family (spouse and children). Regarding religion, 298 participants (48.06%) were orthodox and 37.9% (235) were protestant. A total of 336 (54.19%) participants were married. Over half, 56.61% (351), of participants had had formal education and 40.65% (252) respondents were financially supported by their family, as shown in Regarding social support, (259) 41.77% had moderate social support and (196) 31.61% had poor social support. Regarding serious life events, (243) 39.2% of respondents had experienced serious life events in the previous 6 months. Regarding participant substance use, 45.48% (282) reported ever having used alcohol and 34.68% (215) were current alcohol users, as shown below in The majority, 609 (98.2%), of the respondents had no known history of mental illness, whereas 241 (38.87%) had a history of known chronic disease. Of the total study participants, 128 (20.64%) and 61 (9.83%) had suicidal ideation and attempts, respectively. More than one-third, 218 (35.16%), of the respondents were found to have anxiety. The majority, 603 (97.26%), of subjects had no family history of mental illness .The findings of this study revealed that the prevalence of depressive symptoms among older adults was 51.77% (95% CI: 47.83–55.69%).P-value of less than 0.2. These variables fulfill the minimum requirements for further multivariate logistic analysis. Among these variables, advanced age, being a woman, living alone, having known chronic disease, having anxiety, and having poor social support were statistically significant for depressive symptoms in multivariable analysis, with a p-value less than 0.05.In bivariate logistic regression analysis, variables such as age, being a woman, being divorced, being single, being widowed, source of income, living alone, ever having and recently having used alcohol, having known chronic disease, having attempted suicide, having anxiety, having and poor social support had a In this study, the odds of having depressive symptoms among women were approximately 2.31 times higher compared to men . As age advanced, the odds of having depressive symptoms increased, and elderly individuals who were 70–79 years of age were 1.92 times  more likely to develop depression than those 60–69 years old, whereas those aged 80–89 years were 2.15 times  more likely, and those aged 90 and older were 3.77 times  more likely. In this study, the odds of having depression among elderly participants who lived alone were two times higher than among participants living with family .The result of this study revealed that elderly respondents who had known chronic disease were 3.2 times more likely to develop depression compared to those who had no known chronic disease . The odds of having depression among elderly respondents who had anxiety were 3.4 times higher than those who had no anxiety . Social support was another factor associated with elderly depression. Participants with poor social support were 3.56 times more likely to have depression compared to participants with strong social support , as shown in The findings of this study revealed that the prevalence of depression among elderly people was 51.77%, with a 95% CI: 47.83–55.69. We found that being a woman, being of advanced age, living alone, having a chronic disease, having anxiety, and having poor social support were the associated factors of depression in this study. Regarding the prevalence of depression, the finding of the current study is in line with studies conducted in Nepal, 53.1% ; South AIn the Dega Damot and Wenberema study, a possible reason for the discrepancy might be that the study setting was a rural residence, as well as the differences in sampling techniques used and sample sizes. In the Ghana study, the discrepancy might be attributable to the sample size used, which was 262, and sociocultural differences. This study is also higher than community-based studies conducted in Nigeria, 44.7% ; Nepal, On the other hand, the findings of this study were higher than studies conducted in Thailand, 28.5% ; rural VOur study was also higher than studies carried out in Malaysia 27.3% , Italy 2In the present study, a multivariate logistic regression model revealed that being a woman, being of advanced age, living alone, having a chronic illness, having anxiety, and having poor social support were statistically significant factors associated with depression among the elderly. In this study, women were approximately 2.31 times more likely to have depression than men; this finding was supported by studies conducted in Egypt , India , Brazil The current study showed that, as age advanced, the odds of having depression increased, and those aged 70–79 years were 1.92 times  more likely to develop depression than those aged 60–69 years old, whereas those aged 80–89 years were 2.15 times  more likely, and those aged 90 and older were 3.77 times  more likely. A possible justification for this finding might be that as age advances, people tend to experience reduced daily functioning abilities, a decreased sense of self-worth, a lower degree of life satisfaction, pain, a lower economic status, and familial losses, which can increase the risk of depression. This was in line with studies conducted in Nigeria , VietnamIn addition to this, participants with poor social support were 3.56 times more likely to have depression compared to those with strong social support . This might be due to the fact that elderly people have fewer social connections as a result of illness and disability, which leads them to lose intimate ties and their role in society. This could raise the likelihood of elderly people experiencing depression. This is also supported by studies conducted in Morocco  and WembIn this study, elderly respondents who had known chronic disease were 3.2 times more likely to develop depression compared to those who had no known chronic disease . This may be explained by the fact that emotional instability or depression may be more likely to develop in those who are physically ill than in those who are relatively healthy. A chronic illness is identified as one of the risk factors for depression development in a WHO report. This is in line with studies carried out in Brazil , Sri LanThe findings of our study quantify the burden of depressive symptoms for the benefit of readers, mental health institutions, and policymakers. Furthermore, the results of this study provide information regarding important risk factors for health professionals to design appropriate solutions for the problem, which can improve the mental and physical wellbeing of the elderly population.In the present study, depression was shown to affect more than half of the elderly. Advanced age, being a female, living alone, having a chronic illness, having anxiety, and having poor social support were all strongly associated with depression in the elderly. These results emphasize the need to integrate community healthcare services into counseling and psychiatric services. A thorough screening technique to ensure the early identification of depression among the elderly with medical comorbidity is critical, as it contributes to reducing the illness burden on patients’ families and society as a whole.This study offers valuable information for mental health institutions and policymakers interested in enhancing the health and quality of life of the elderly population. We recommend that future researchers consider the assessment of the cognitive status of the elderly population. The cross-sectional nature of the study design, however, limits making firm conclusions on the link between geriatric depression and risk variables.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by University of Gondar Ethical Review Board. The patients/participants provided their written informed consent to participate in this study.AM, TA, YM, SK, GN, AK, JD, NB, DD, and HA participated in the inception of the idea, proposal development, data collection, analysis, and final write-up. All authors approved the final manuscript.


Cancer remains an enduring challenge in modern society, prompting relentless pursuits to confront its complexities. However, resistance often emerges against conventional treatments, driven by their inherent limitations such as adverse effects and limited solubility. Herein, we spotlight a remarkable solution; a niosomal platform engineered to tandemly ferry two potent agents, doxorubicin (DOX) and curcumin (CUR). Notably, we delve into the pivotal role of PEGylation, unraveling its impact on therapeutic efficacy. These niosomes consist of Span 60, Tween 60, and cholesterol with a molar ratio of 5:2:3, which were prepared via a thin film hydration method. The physicochemical characterization of particles was performed using DLS, zeta potential measurement, SEM, and FTIR analysis. In addition, their encapsulation efficiency and release profile were determined using the HPLC method. Finally, their cytotoxicity and biocompatibility effects were checked by performing an MTT assay test on the MCF7 and L929 cell lines. The obtained results confirmed the successful fabrication of co-loaded niosomal structures with and without PEG coating. The fabricated nanoparticles had sizes in the range of 100 to 200 nm with a surface charge of about −18 mV for particles without PEG coating and −40 mV for coated particles. Notably, DOX encapsulation efficiency leaps from 20% to 62% in the transition from uncoated to coated, while CUR exhibits an impressive surge from 80% to 95%. The drug release was more controlled and slower in the coated sample. Finally, the MTT results confirmed the biocompatibility and synergistic effect of the simultaneous use of two drugs on cancer cells in the PEGylated niosomal particle. Based on the results, PEGylated niosomal particles can be considered adept vehicles for the simultaneous delivery of different chemotherapy cargoes with synergic interaction to overcome cancer. The goal of most cancer treatments is to limit tumor cell proliferation and prevent metastasis . NeverthIn recent years, nanotechnology, via creating ideal carriers with biocompatibility properties, capable of carrying a high dose of medicine, and stability in blood circulation, has attracted lots of attention . NiosomeThey deliver therapeutic compounds to the intended site and reduce the side effects on normal tissues and cells . To overThe aim of this research is to fabricate a type of niosomal formulation that contains both hydrophobic and hydrophilic therapeutic compounds, simultaneously, for cancer treatment. In addition, we have compared the effectiveness of PEGylation on different features of the fabricated niosomes. To reach these aims, drug-loaded niosomal formulations were fabricated via the thin-film hydration method, and the physiochemical properties of both coated and noncoated systems, in terms of stability, drug loading, drug release, morphology, size, and surface charge, were detected and compared. Additionally, their biological activity was studied to check their cytotoxicity performance on both healthy and cancerous cell lines.Sorbitan monostearate 60 , polyoxyethylene-sorbitan-monostearate , cholesterol , Polyethylene Glycol 2000 (PEG2000), Trifluoroacetic acid (TFA), Acetonitrile, Dulbecco’s modified Eagle’s medium (DMEM) including 10% fetal bovine serum (FBS), thiazolyl blue tetrazolium bromide (MTT) powder, phosphate-buffered saline salts (PBS), and dimethylsulfoxide (DMSO) were purchased from Sigma, St. Louis, MO, USA. Isopropyl alcohol (≥99.9%), chloroform (≥99.9%), and ethanol (≥99.9%) were bought from Merck, Darmstadt, Germany. Curcumin (≥99%) and doxorubicin hydrochloride  were purchased from Actore Co, Karaj, Iran. In addition, ThermoFisher Scientific Inc,  provided penicillin/streptomycin (0.5%) and trypsin/EDTA (0.5%).For the fabrication of noisome, Span 60, Tween 60, and cholesterol with a molar ratio of about 5:2:3 along with 5 mg of curcumin were dissolved in 10 mL of chloroform and stirred for 15 min. Then, the organic phase of this solution was removed using a rotary evaporator  at 45 °C with an approximate speed of 150 rpm to form a thin lipid film . To gene−1. The size and surface charge of niosomes made with one drug and two drugs simultaneously before and after coating with polymer were investigated by dynamic light scattering (DLS) and zeta potential analysis . The morphology of the resultant niosomes was determined by scanning electron microscopy .Surface functional groups of the niosomes underwent scrutiny via infrared spectroscopic analysis , encompassing the spectral range of 400–4000 cm®, Berlin, Germany) method was used to determine the exact amount of two drugs entrapped in the niosomal formulation. Briefly, 40 µL of the supernatant was added to the Eurospher II column 100-5 C18 4.6 × 250 mm over the period of 20 min and a controlled flow rate of 1 mL/min. Notably, absorbance measurements occurred at the wavelengths of 250 and 485 nm for doxorubicin, and 420 nm for curcumin. The mobile phase encompassed a balanced blend of methanol and water, infused with 0.2% trifluoroacetic acid (TFA) and acetonitrile, orchestrated with diverse molar ratios as delineated in To quantify the encapsulated quantities of DOX and CUR within the niosomes, 300 µL of drug-loaded niosomal suspension was mixed with 2700 µL of 2-propanol 1% w/v solution in water, and the solution was shaken for 48 h at room temperature. Then, it was centrifuged for 10 min at 10,000 RPM and the supernatant was separated. Next, the high-performance liquid chromatography :After 4 months of storage of the fabricated particles at the controlled temperature of 4 °C, three analyses of DLS, SEM, and encapsulation efficiency measurement were repeated on both PEGylated and non-PEGylated co-loaded niosomes to track their changes during the storage time.To elucidate the release pattern of niosomes (with/without coating), drug-loaded particles were dispersed in two different pHs of 6.6 and 7.4 PBS, which simulated the pH of cancerous and normal tissue, respectively. Suspension of drug-loaded samples was put into dialysis bags (12 kDa) and then immersed in PBS solutions (with pH 6.6 and 7.4) and then incubated for 10 days at 37 °C. At designated intervals , discreet samples were extracted from the outer solution, and the amounts of the released drug were determined via HPLC analyses using 420 nm and 480 nm wavelengths for DOX and curcumin, respectively.4 cells of each cell line were implanted in each well of 96-well plates undergoing a 24 h incubation period at 37 °C with 5% CO2. Next, the medium of each well was replaced with 200 µL of fresh one, including various treatments with two concentrations (10 µg/mL and 80 µg/mL). Fresh media and media containing free concentrations of drugs were used as negative and positive controls, respectively. The treated cells were incubated for 48 h at 37 °C. After that, cells were washed with PBS (twice) and then incubated for 4 h with a 100 µL culture medium containing MTT solution (5 mg/mL). In the next step, the medium of each well was replaced by 100 µL DMSO and incubated for an hour in the dark, followed by reading the absorbance of the wells with an ELISA reader  at 570 nm. In addition, the pivotal parameter of inhibitory concentration (IC50) values was deduced for each treatment category employing Equation (2). This value serves as a cardinal metric, elucidating the concentration of treatment requisite to curtail 50% of cell vitality.The evaluation of cytotoxicity was undertaken employing two distinct cellular entities: the human breast cancer cell line (MCF-7) and the healthy mouse fibroblast cell line (L-929). In brief, 1 × 10This value represents doxorubicin IC50 when combined with curcumin at concentration b, and A represents dox IC50 when used alone, while B represents curcumin IC50 when used individually. The numerical value of the Combination Index (CI) stands as a pivotal indicator, deciphering the interplay between the two compounds; a value higher than 1 indicates antagonistic interaction and a value of 1 indicates additive interaction ,17,18.p value ≤ 0.05 was used for MTT experiments.To evaluate the significance of quantitative data, SPSS software ) was used. It is noticeable that the The size of niosomes containing DOX or CUR (DOX loaded niosome and CUR loaded niosome), and the combination of them (Co-loaded niosome) before and after coating with PEG2000 polymers was investigated using DLS analysis . As can Additionally, Zeta potential analysis was performed to determine the surface charge of the particles before and after PEGylation. Actually, the surface charge of the particles can affect their interaction with each other (which could lead to aggregation of the particles) and also with the cellular membrane (which could affect their cellular uptake). Therefore it is important to measure the surface charge of the particles. As is shown in −1, which is attributed to the C-O vibrating bond of cyclohexane in Span 60 and cholesterol. Additionally, bands at around 1730 cm−1 and 2929 cm−1 were related to the C = O stretching vibration bond of ester groups in Span 60 or Tween 60 and the C-H bond of the carboxylic group in cholesterol, respectively [−1 was seen in all components that were attributed to the O-H bond of the hydroxyl group [−1 and 940–1100 cm−1 were observed in co-drug-loaded particles, which are attributed to the N- H and C = C bonds of the CUR and DOX drugs, respectively [To evaluate the interactions between the niosomal components, FTIR analysis was performed a. The obectively . In addiyl group . Figure ectively .Scanning electron microscopy (SEM) analysis was performed to evaluate the morphology of DOX and CUR co-loaded niosomes with and without PEG-coating. As is shown in Quantitative elucidation of encapsulation efficiency (EE%) for DOX and CUR within the niosomal carriers was meticulously pursued via the HPLC method. The results of this study  showed ETo evaluate the effect of PEGylation on the stability of niosomal particles during the time, both co-loaded particles in coated and non-coated forms were kept at 4 °C for 4 months, and then their size and morphology, as well as EE%, were measured. Based on the DLS results, the size of coated and noncoated particles changed to 306.4 nm and 499.2 nm, respectively. Furthermore, the SEM images provided significant aggregation between the particles without the PEG layer a, while 50 values of different types of treatments are presented in To evaluate the survival rate of the cells, MTT assay was used against MCF-7 and L929 cell lines as cancerous and normal cell lines, respectively. The results demonstrated that the drug-loaded niosomal systems reduce the growth of MCF-7 cells in a concentration-dependent manner . Here, 1The effect of the synthesized nanoniosomes was also investigated on the normal cell line of L929 c,d. The In this study, two drugs (curcumin and doxorubicin) were simultaneously loaded in niosomal nanocarriers to improve their therapeutic effect and reduce their side effects during chemotherapy usage. Moreover, a comparison study was performed between PEGylated and nonPEGylated co-loaded niosomes. In detail, six types of niosome were prepared using Span 60/Tween 60/cholesterol with different drug contents in the form of individual drug-loaded and co-loaded with and without PEG coating on their surface. Additionally, their characteristics were evaluated using several analyses including DLS, FTIR, zeta potential, and SEM. The obtained results showed that the size of the particles was between 150 and 200 nm for the noncoated category, and betwen 200 and 300 nm for the coated ones, which can be attributed to the placement of PEG branches on the surface of the carriers ,28. It iIn the next step, the bioactivity of the fabricated nanoparticles was assessed. For this goal, the different types of niosomal particles were investigated in terms of EE%, stability, release patterns, and cytotoxicity. The achieved data from measuring the EE% of the loaded DOX showed that after PEGylation, its EE% increased dramatically from approximately 20% to 80%, which can be attributed to the trapping of DOX molecules between the PEG polymer branches on the surface of the particles . FurtherMoreover, the evaluation of the stability of NPs demonstrated similar results compared to the studies by Z.liu et al.  and SeleAdditionally, the therapeutic effect of the particles was evaluated via MTT assay on the cancerous MCF-7 cell line and normal L929 cell line. Our outputs presented that co-loading of DOX and CUR in niosomal carriers enhanced their therapeutic potency in comparison to the single-form usage of DOX or CUR, agreeing with the study conducted by Firouzi et al. . In addiThe purpose of this study was to elevate the therapeutic potential of both DOX and CUR via their dual incorporation within niosomal carriers and improvement of the niosomes’ properties with the help of PEGylation on their surface. The desired NPs were fabricated successfully with a size of about 275 nm and EE% of 80% and 90% for DOX and CUR, respectively. They showed a more controllable release manner and enhanced cytotoxicity on cancer cells after PEGylation. The results confirmed that the strategy of co-loading DOX and CUR led to a synergic therapeutic effect against the cancer cells. In addition, PEGylation successfully promoted the therapeutic features, stability, and biocompatibility of the particles. So, it could be concluded that the strategy of usage of this co-loaded PEGylated niosomal particles can be considered a suitable cancer treatment.


Objective: Pancreatic adenocarcinoma (PAAD) is a highly malignant gastrointestinal tumor with almost similar morbidity and mortality. In this study, based on bioinformatics, we investigated the role of gene methylation in PAAD, evaluated relevant factors affecting patient prognosis, screened potential anti-cancer small molecule drugs, and constructed a prediction model to assess the prognosis of PAAD.Methods: Clinical and genomic data of PAAD were collected from the Tumor Genome Atlas Project (TCGA) database and gene expression profiles were obtained from the GTEX database. Analysis of differentially methylated genes (DMGs) and significantly differentially expressed genes (DEGs) was performed on tumorous samples with KRAS wild-type and normal samples using the “limma” package and combined analysis. We selected factors significantly associated with survival from the significantly differentially methylated and expressed genes (DMEGs), and their fitting into a relatively streamlined prognostic model was validated separately from the internal training and test sets and the external ICGC database to show the robustness of the model.Results: In the TCGA database, 2,630 DMGs were identified, with the largest gap between DMGs in the gene body and TSS200 region. 318 DEGs were screened, and the enrichment analysis of DMGs and DEGs was taken to intersect DMEGs, showing that the DMEGs were mainly related to Olfactory transduction, natural killer cell mediated cytotoxicity pathway, and Cytokine -cytokine receptor interaction. DMEGs were able to distinguish well between PAAD and paraneoplastic tissues. Through techniques such as drug database and molecular docking, we screened a total of 10 potential oncogenic small molecule compounds, among which felbamate was the most likely target drug for PAAD. We constructed a risk model through combining three DMEGs  with clinical factors significantly associated with prognosis, and confirmed the model robustness using external and internal validation.Conclusion: The classification model based on DMEGs was able to accurately separate normal samples from tumor samples and find potential anti-PAAD drugs by performing gene-drug interactions on DrugBank. Pancreatic adenocarcinoma (PAAD) is the 14th most common cancer globally , with anEpigenetics are changes in gene expression but not in DNA sequence, and the major epigenetic alteration leading to PAAD progression is DNA methylation . To deteAs research continues, aberrant methylation of DNA CpG islands has become a prominent feature of PAAD and a potential diagnostic marker and therapeutic target for PAAD. However, the results of clinical trials were disappointing, probably due to the low level of epigenetic specificity . Therefohttps://portal.gdc.cancer.gov/) by means of UCSC Xena. The gene expression profiles of normal pancreas samples were obtained from the GTEX  databases using UCSC Xena.Methylation data, clinical follow-up data, and gene expression profiles of PPAD came from TCGA  of the RefSeq genes. For each probe, the raw gene methylation intensity was expressed as a beta value. To identify differentially methylated CpG sites (DMS), PAAD tumor samples were compared with paracancer samples using the “limma” R package   . Statisthttps://www.illumina.com/). In different regions , the average β-values of genes were calculated with the correspondence. Using the “limma” R package, the differentially methylated regions were calculated, where FDR <0.01, delta β-values < -0.1 were the demethylated regions, FDR <0.01, delta β > 0.1 were hypermethylated regions.The CpG locus to gene match files were downloaded from the Illumina website  method, where FDR >0.01 and log2FC > 2 were up-regulated genes, and FDR >0.01 and log2FC < −2 were down-regulated genes.Differentially expressed genes (DEGs) were analyzed for normal and tumor samples in the TCGA-PAAD cohort using the “limma” R package, and To identify the relationship between gene methylation and gene expression profiles, we took the intersection of differentially methylated genes and DEGs to obtain differentially methylated and expressed genes (DMEGs) and classified them into four groups: HyperDown, HyperUp, HypoDown, HypoUp . Then, wDEGs were proposed as tumor markers for the diagnosis of PAAD, and 50% of the expression profile data of DMEGs and methylation data of DMEGs for PAAD were the training set and 50% as the test set. The training set data were analyzed by principal component analysis (PCA) with the “prcomp” R function  to clarihttps://go.drugbank.com/) and the Protein-Protein interaction (PPI) network (threshold score of 400). Here, given distance d  as the shortest path between node s and node t , D (degree of related gene set nodes in PPI), T (set of drug target genes), S (PAAD-related genes), and the calculation is as follow:ω, the weight of the target gene, was calculated as ω = -ln (D+1) if the target gene was a gene in the PAAD-related gene set, otherwise ω = 0.The use of key genes as potential therapeutic targets is a cornerstone in the development of therapeutic agents for sepsis. We determined PAAD and drug proximity based on drug-target pairs from the drugbank database , and generated the simulated reference distributions after performing random repetitions for 10,000 times, at the same time we the observed distances corresponding to the actual were scored using the mean and standard deviation of the μd  and σd  reference distributions and converted into a normalized scoring, i.e., the proximity z.A technique for designing drugs based on receptor features and the way that drug molecules interact with receptors is called molecular docking. In the realm of computer-aided drug development, it is a theoretical modeling technique that primarily investigates the interaction between molecules (such as ligands and receptors) and forecasts their binding mechanism and affinities. . AutodocIn this study, the binding stability of the receptor-ligand complex was assessed by performing molecular dynamics simulations of 100 ns  using thIn the TCGA-PAAD dataset, we first randomly and equally divided 241 KRAS wild samples into training (Train) and validation (Test) groups according to the ratio of 1:1, and then reduced the associated genes  by LeastTo verify the robustness of the gene signature model, we first used the same model and the same coefficients as the training set in the validation set, and then compared the survival differences between the two groups by log-rank test. After that, we downloaded the expression profiles of PAAD from the ICGC database as well as clinical information, and then used the model constructed above to calculate each score separately and obtain the best cutoff, and then performed the survival curve analysis in the external dataset for the high- and low-risk groups.https://cran.r-project.org/doc/manuals/R-lang.html). The optimal threshold of gene expression or score was selected for risk grouping of PAAD using the surv_cutpoint function of the “survminer” package. The Kaplan-Meier assessment method was used to assess the survival differences between the low-risk and high-risk groups, and the Log-rank test was used for comparison. Unless otherwise stated, all statistical tests were two-sided and p < 0.05 was considered statistically significant. Comparisons between multiple groups were performed and plotted using the “ggpubr” and “ggplot2” packages, and the statistical significance of box plots was assessed using the Mann-WhitneyU or Kruskal–Wallis tests.All statistical analyses were operated in R software  were obtained by intersection analysis of DMGs and DEGs. In Gene body, TSS200 and TSS1500, 141, 187 and 154 DEMGs were obtained, respectively . The metTo further investigate the role of DMEGs in PAAD, we first used the “circlize” package to map the distribution of 318 DMEGs on chromosomes, with chromosomes chr11 and chr12 having the largest number of 26 DMEGs, chr10, chr12, chr17, chr16, chr2, chr19, chr3, chr20, chr5, chr4, chr7, chr6, and chr6. Chr17, chr16, chr2, chr19, chr3, chr20, chr5, chr4, chr8, chr7, chr6 chromosomes also possessed more than 10 DMEGs each . We consAs mentioned previously, DMEGs may be the key genes causing PAAD, and therefore targeting DMEGs is a potential target for the treatment of PAAD. To this end, we calculated the proximity of DMEGs to PAAD according to https://www.alphafold.ebi.ac.uk/) website for ADRA1A homology modeling to obtain the 3D structure of the ADRA1A protein and the Deepsite (https://www.playmolecule.com/deepsite/) website to predict the protein activity of ADRA1A (32). In addition, the Gromacs2019 software package was used to predict potential small molecule compounds, and a total of 10 small molecule compounds were identified by calculating RMSD values, namely DB06201, DB12733, DB00610, DB00450, DB00699, DB06706, DB06711, DB06764 DB00949, and DB08954  9 had the highest molecular docking score and therefore had a higher potential to be a potential inhibitor of GRIN2B protein. Compound DB0094 interacted with GRIN2B protein, and the RMSD value of compound DB0094 was relatively stable overall  . The comn = 121) and one as the validation set (n = 120). We used 10-fold cross-validation to execute 1,000 Lasso regression analysis on the expression and clinical survival data of these 318 DMEGs genes, and we counted the appearances of each probe 100 times . Then, we used the “timeROC” package to perform ROC analysis for prognostic classification of RiskScore, and the AUCs of predictive classification efficiency were 0.82, 0.89, and 0.77 for one-, three-, and five-year, respectively (p < 0.0001).According to the expression level of the sample, we calculated the risk score for PAAD samples, and the RiskScore distribution is shown in ectively , suggestp = 0.00039, HR = 3.78). Finally, we performed the same validation in the ICGC-PAAD external data cohort, and the RiskScore distributions for each sample are shown in p = 0.00024, HR = 1.68).The model was validated further by using the same coefficients and model in the training set as in the validation set. The risk score of each sample was calculated using the same method, and the RiskScore distribution is shown in PAAD as one of the most lethal and aggressive malignancies has a 5-year survival rate of less than 10%, , and is In the early 20th century, Fukushima N and other scholars extensively studied the methylation of different genes in PAAD and its precancerous lesions , and found abnormal methylation of ppENK and p16 (13). Next, it was shown that the incidence of aberrant methylation was 7.3%–7.7% in PanIN-1 patients, 22.7% in PanIN-2 patients, and 46.2% in PanIN-3 patients, a phenomenon that suggests that the incidence of aberrant methylation increases with a more advanced PanIN grade, but the exact mechanism is not clear . Our stuIllumina human methylation 450 k bead array provides a better technical platform for further study of DNA methylation, therefore, we focused on methylation genes within the three regions of Gene body, TSS1500, and TSS200. A total of 758 hypermethylated genes and 418 demethylated genes were identified within the Gene body region, which was consistent with the incidence of PAAD hypomethylation reported in previous studies, and hypomethylation was mainly associated with cell cycle cycling, cell differentiation, and cell surface antigen/cell adhesion . TSS1500In 2005, the European Palliative Care Research Collaborative (EPCRC) network working group screened important clinical markers for survival prediction in patients with end-stage cancer based on decades of clinical evidence and recommended a variety of prognostic tools. On this basis, researchers have successively validated and derived several relevant prediction models according to cancer types, and PAAD prognostic models have emerged, which can be broadly classified into traditional manual prediction and statistical-based bioinformatics modeling, with the latter being the majority at present, but they all share common problems such as small sample size, low specificity, and poor predictive performance  Wang et. CompareThere are some limitations in this study. Although the results showed that 3-DMEGs-based signature could distinguish tumor samples and normal samples, the model reliability should be improved with long-term clinical application. Additionally, we downloaded expression profiles and methylation data of PAAD from public databases. Thus, further prospective data should be collected to validate the results. Besides, experimental studies and clinical trials should be performed to verify the results of molecular docking in this study.With the gene expression profile data of PAAD, we identified DEGs and DMGs between normal samples and tumor samples with KRAS wild type; the classification model based on DMEGs was able to accurately separate normal samples from tumor samples, and the gene-drug interactions were performed on DrugBank to find some potential anti PAAD drugs.


Zinc phosphide is a rodenticide that is used in agricultural, urban and industrial environments in México. After ingestion, it reacts with hydrochloric acid, hydrolyzing into phosphine. It causes cellular hypoxia via mitochondrial toxicity, resulting in multiple organ dysfunction and death. There is no antidote or specific treatment for zinc phosphide toxicity. We present the case of a 45-year-old female who ingested zinc phosphide with suicidal intent. On arrival at the emergency department, she had multisystemic disorders. Supportive care, decontamination and antidotal therapy were initiated. Subsequently, she evolved to clinical improvement with a resolution of the biochemical abnormalities of tissue hypoperfusion. She was discharged on day 7 without complications. In this review, we provide updated therapeutic options and discuss their specific pathophysiological basis. Zinc phosphide is a rodenticide that has been used since 1940 in agricultural, urban and industrial environments . Since 1When zinc phosphide comes into contact with water or acid and undergoes hydrolysis, it releases phosphine gas, which is the active component ,2,6,7,8.We present the clinical case of a 45-year-old woman who ingested zinc phosphide with suicidal intent. An analysis of the therapeutics used and a review of the scientific literature on the subject are presented.2 87% (FiO2 21%) and a FOUR scale score of 16 points. The clinical toxicology service was notified immediately. Electrocardiogram  0.6, methemoglobin (MetHb) 1.1, HCO3 10.5, EB −14.3 and lactate 7.7.A 45-year-old female with a history of untreated depressive disorder was admitted to the emergency department 6.5 h after ingesting 6 to 9 g of zinc phosphide . She was found at her home in a supine position with indifference to the environment, with the presence of involuntary uresis and emesis of black content. A 50 g container of 24% zinc phosphide was found next to her. Later, she reported that she had consumed 500 cc of beer and, subsequently, three-quarters of the contents of the bottle of zinc phosphide. Upon admission to the emergency department, she was awake and reported nausea, vomiting, epigastric pain and anxiety. Her vital signs were as follows: blood pressure 94/74 mmHg, heart rate 129 beats per minute, respiratory rate 26 per minute, temperature 36.5 °C, SaOrdiogram : sinus tGastric lavage was performed upon admission (7 h post-ingestion) with coconut oil (200 mL) plus bicarbonate (7.5% 50 mL). Continuous cardiac monitoring, request for laboratory studies and administration of crystalloid solutions were performed. She did present metabolic acidosis with a high anion gap with worsening for the first 6 h. The hyperinsulinemia–euglycemia therapy was initiated eleven hours after xenobiotic ingestion , with a Nine hours after the treatment was started, she showed clinical and biochemical improvement , and 16 Zinc phosphide is colorless, odorless, flammable and highly toxic; when it comes into contact with water or hydrochloric acid in the stomach ,14, it qThe mechanisms of toxicity are not clear, but several were described. Cellular hypoxia caused ,14,19 byThe clinical manifestations are due to multi-organ toxicity , secondaThe safety of medical and nursing personnel with adequate personal protective equipment is mandatory given that phosphine gas is released quickly and easily. The appropriate equipment consists of respiratory protection with a full-face mask with positive pressure since the concentration is unknown and mucotegumentary protection with gloves and a gown. Although absorption through the skin and eyes has not been frequently reported ,7,24, thTo decrease the absorption of the xenobiotic in the exposed patient, gastric lavage and activated charcoal are described as effective measures. These should not be done with water because it would favor the formation of phosphine; therefore, the literature reports performing gastric lavage with coconut oil, potassium permanganate  or liquid paraffin, considering that stomach acid stimulates the conversion to phosphine gas ,14. GastIn the clinical case presented, gastric lavage was performed 7 h after ingestion, with a mixture of 200 mL coconut oil (melted in a water bath) and 50 cc 8.4% sodium bicarbonate   mediate the mechanism of phosphine toxicity; therefore, using antioxidants such as N-acetylcysteine, glutathione, melatonin, vitamin C, vitamin E and carotenes  may be bMyocytes are highly susceptible to oxidative stress, which leads to a reduced ejection fraction of the left ventricle and, consequently, to heart failure. Therefore, the correction of electrolyte alterations and the acid–base balance that limits the arrhythmogenic potential must be a priority and immediate . The medInsulin has been known to have positive cardiac inotropic properties since the 1930s, although the mechanism is not fully understood. The use of high doses of insulin together with glycemic support, also known as hyperinsulinemia–euglycemia therapy (HIET), was initially used in toxicology in poisoning beta-blockers and calcium antagonists ,18,45. IFor a toxin such as phosphine, which induces myocardial depression in up to 60% to 100% of cases , the utiIn the absence of stress, the heart primarily catabolizes free fatty acids to obtain its energy needs ,28,33,45HIET provides metabolic support to the alterations associated with cardiotoxic shock . It incrWhen regular insulin is administered intravenously, an immediate peak plasma concentration is obtained. The onset and peak of the cardiovascular effects of insulin occur between 15 to 40 min after administration ,42,47, aRapid-acting insulin was used at a concentration of 10 international units (IU) for every 1 milliliter of 0.9% saline solution , that isThe initial dose of insulin is 1 IU/kg in bolus and the glucose supply is calculated at 0.5 g/kg ,47; if gHemodynamic monitoring should be performed for the cardiac ejection fraction and cardiac contractility ,47. The In the clinical case presented, therapy was started with a bolus of insulin at a dose of 1 IU/kg and a maintenance dose of 1 IU/kg/h. Capillary glucose was monitored every 20 min until the targets were reached and hourly thereafter. The maximum insulin infusion dose was 7 IU/kg/h at 8 h from the start of treatment. Dose reduction began at hour 72 of treatment and was discontinued at 78 h after initiation. A capillary glucose target between 100 and 250 mg/dL was maintained. The glucose supply began at 0.5 g/kg, with a dose adjustment of 0.7 g/kg, and withdrawal was achieved 22 h after stopping the insulin therapy. Indirect biochemical variables of tissue perfusion were measured, such as pH, lactate and bicarbonate, as well as a close clinical evaluation, observing improvement all around .According to the literature, the duration of therapy with high doses of insulin lasts from 9 to 49 h, with an average of 27 h . The witThe following graph  shows thBased on the pattern shown by this patient, the following scheme was proposed as a point of good practice, with capillary glucose monitoring every 2 h. In our case, 5 h after the withdrawal of hyperinsulinemia therapy, the intravenous glucose intake was reduced by 0.1 g/kg. Six hours later, it was reduced again by 0.1 g/kg, and 9 h later, it was reduced again by 0.1 g/kg. After 20 h post discontinuation of high-dose insulin, it was reduced by 0.1 g/kg/hour per hour, achieving cessation at 22 h after the initiation of withdrawal.Intravenous lipid emulsion (ILE) was introduced in 1960 as an energy substrate because it contains essential fatty acids, and it was administered parenterally as a nutritional supplement in seriously ill patients ,52. Its 2+ homeostasis and rescue signaling pathways that regulate the opening of the mitochondrial permeability transition pore (mPTP), which restores mitochondrial Ca2+ levels [ILE’s mechanism of action in phosphine intoxication is not defined, and neither is its mechanism of action with some water-soluble xenobiotics . However+ levels .A series of cases of aluminum phosphide poisoning were published in countries such as India ,60,61,62The clinical and biochemical response showed improvement with ILE 20% ; at the A range of options were described for when cardiotoxic shock occurs, such as:Digoxin, which would increase cardiac contractility and blood pressure, at a dose of 0.5 mg every 6 h for one day and subsequently 0.25 mg every 24 h ,65.Trimetazidine, which inhibits 3-ketoacyl CoA thiolase in the mitochondrial matrix and catalyzes fatty acids into acetyl CoA for its entry into the Krebs cycle and consequent production of ATP, thus preventing the decrease in intracellular ATP. It inhibits the oxidation of fatty acids and favors the oxidation of glucose, ensuring the functioning of ionic pumps and the transmembrane flow of sodium and potassium, and maintaining cell homeostasis. The suggested dose is 20 mg to 35 mg every 12 h ,14.Melatonin has an amphiphilic structure, which penetrates the mitochondria. Various benefits are described for it: it prevents apoptosis by restricting mitochondrial pore permeability and disturbing caspase activation, intensifies the production of ATP, weakens the suppression of the respiratory chain and it has antioxidant effects.Levothyroxine decreases the activity of caspases 3 and 9, increasing cell viability, at a recommended dose of 3 mcg/kg.Taurine, which enhances the detrimental effects of phosphine on the electron transport chain, enhances mitochondrial membrane potential and reduces oxidative stress.Boric acid, which is recognized as a Lewis acid, and thus, it has an empty p orbital, which can accept electrons, and phosphine offers electrons to cytochrome C oxidase, interfering with the mitochondrial respiratory chain; therefore, phosphine reacts with boric acid and produces a gaseous adduct, where the frequency of gas evolution decreases but not the volume of release.Other drugs with an antioxidant function are as follows:Vitamin E at a dose of 100 U every 12 h for 72 h.Vitamin C at a dose of 1 g every 8 h in the first 24 h.However, neither of these have a routine recommendation .In this case, the therapeutic approach was decided in a stepwise manner due to the lack of clinical improvement and the worsening of tissue hypoperfusion biomarkers during the first hours of management.From the beginning, management with antioxidant therapies and electrolyte support with magnesium was indicated. N-acetylcysteine was administered to protect against fostin-induced myocardial oxidative damage and to restore intracellular glutathione levels. Likewise, with the aim of preventing hepatic necrosis, electrolyte support with magnesium sulfate was initiated from the beginning, considering that phosphine induces oxidative stress, which increases lipid peroxidation and reduces glutathione levels, with a consequent decrease in magnesium and magnesium-dependent glutathione. The literature reports a mortality reduction effect of over 50% using this strategy, which indicates its importance. Its effects are synergistic with N-acetylcysteine in restoring glutathione and magnesium-dependent glutathione levels. At the same time, management with HIET was started, providing metabolic and cardiovascular support. Considering its vasodilator effect at the coronary level, it prevents myocardial hypoperfusion and progression to cardiotoxic shock. After the first 3 h of HIET, an increase in lactate from an initial value of 7.7 to 8.4 mmol/L was observed, accompanied by a decrease in pH from 7.29 to 7.22, as well as a decrease in serum bicarbonate from 10.5 to 5.1 mmol/L. Furthermore, at the clinical level, a drop in mean blood pressure from 93 to 76 mmHg was observed. Therefore, ILE was indicated as a second antidotal line to provide a means of redistribution of the xenobiotic and reducing its toxic effects, and thus, provide stability to the mitochondrial membrane and maintain ATP production in the myocardium.Two and a half hours after initiation, the pH increased from 7.22 to 7.26, bicarbonate increased from 5.1 to 9.8 mmol/L and serum lactate dropped from 8.4 to 5.6. Ten hours after the start of ILE, a pH of 7.42, bicarbonate of 17.2 and lactate of 2.4 were observed. During the evolution, no vasopressor support was required and no evidence of cardiac failure was observed. Moreover, bicarbonate replacement was not required.2+ homeostasis and rescue signaling pathways that regulate the opening of the mitochondrial permeability transition pore (mPTP), which restores mitochondrial Ca2+ levels, i.e., a synergistic effect between the inotropic effects of HIET and ILE was contemplated. Although the broader recommendations in the medical literature support the use of regular insulin to institute HIET therapy, it was noted in this case that the use of rapid-acting insulin is equally safe and effective.This behavior supports the theory that the vasoregulatory and inotropic effects of insulin in conjunction with the hyperglycemic substrate of HIET provide metabolic support to the myocardium, preventing it from developing cardiotoxic shock, even in this case in which the ingested dose exceeded the proposed lethal dose 50 for humans. Regarding the possible positive effects of the use of ILE in this case, it was contemplated that the cardioprotective action of long-chain fatty acids in ILE involves CaTaking this into consideration, the following therapeutic proposal was made. We suggest early initiation of support with magnesium sulfate and N-acetylcysteine, together with EILH and ILE in a staggered manner. The use of vasopressors and inotropic support should be reserved only for the case of evolution to cardiotoxic shock .Zinc phosphide poisoning is highly lethal and there is no specific antidote; even the literature is controversial. It can occur accidentally or voluntarily due to suicidal intentions. Despite the lack of international guidelines for its care, the understanding of its pathophysiological bases allows for an adequate approach that, through current therapeutic strategies, not only allows for increasing survival but also opens the door to further research in terms of both the mechanisms of action of each therapeutic resource and the development of better therapeutic options. Herein, we present a case of ingestion of zinc phosphide with suicidal intent at a dose above the LD50, which responded adequately to our stepwise therapy proposal in which early initiation of therapy surely increased survival.The first step for the first-contact physician is not to fear exposure since there is adequate personal protective equipment to provide immediate and high-quality care; therefore, emergency medical services must have the necessary material and human resources.