Results demonstrated the association between DNA damage levels and a decrease in cell proliferation with anthropometric measurements and the severity of obese status, together with elevated levels of urates, inorganic phosphates, chlorides, and hs troponin I levels. DII was connected with oxidative DNA damage, while BMI and basal metabolic rate (BMR) were associated with a decrease in cell proliferation and DNA damage creation. Measured daily BMR and calculated daily energy intake from the food frequency questionnaire (FFQ) demonstrated no significant difference . Groups with higher DNA damage than expected , consumed daily, weekly, and monthly more often some type of food groups, but differences did not show a clear influence on the elevated DNA damage levels. Combination of all three DNA damage assays demonstrated that some type of damage can start earlier in the obese individual lifespan, such as nuclear buds and nucleoplasmic bridges, then comes decrease in cell proliferation and then elevated micronucleus frequencies, and that primary DNA damage is not maybe crucial in the overweight, but in severely obese. Biochemically changed parameters pointed out that obesity can have an impact on changes in blood cell counts and division and also on genomic instability. Assays were able to demonstrate groups of sensitive individuals that should be further monitored for genomic instability and cancer prevention, especially when obesity is already connected with comorbidities, 13 different cancers, and a higher mortality risk with 7–10 disease-free years loss. In the future, both DNA damage and biochemical parameters should be combined with anthropometric ones for further obese monitoring, better insight into biological changes in the severely obese, and a more individual approach in therapy and treatment. Patients should also get a proper education about the foodstuff with pro- and anti-inflammatory effect.Although obesity with its comorbidities is linked with higher cancer risk, the data on genome stability in the obese/severely obese are scarce. This is the first study with three DNA damage assessment assays (Fpg-modified and alkaline comet assays and micronucleus  AmonIn detail, nutritional components for the calculation of inflammatory dietary indices were taken from databases containing the chemical composition of food and beverages. Depending on the method of preparation, the values for flavan-3-ols, flavones, flavonols, flavonones, anthocyanidins, and isoflavones in raw foods were multiplied by retention factors. The retention factor for cooking is 0.59, for frying 0.5, and for baking 1.09 . For eacAll participants were measured for their height with the stadiometer. Another anthropometric analysis was performed with a body composition analyzer  according to the manufacturer’s instructions. Analysis was performed by the medical personnel in the hospital/Institute for Public Health mentioned for volunteer recruitment on the same day as the blood sampling.−1, obesity degree, recommended calorie intake per day), and InBody score.The analyzer gave information about body composition , muscle-fat analysis , obesity analysis (body mass index—BMI and percentage of body fat -PBF in %), segmental lean analysis, segmental fat analysis, weight control (kg), waist-hip ratio (W-H ratio), visceral fat level (VFL), and research parameters  and for DNA damage assessment (heparin tubes and EDTA tubes) .−1) × insulin (mIU L−1)/22.5. An automated hematology analyzer (XS-1000i) with an autosampler  was used to determine the complete blood count (CBC).The clinical chemistry autoanalyzer Beckman Coulter AU 480  was used to determine the levels of glucose, urea, urates, inorganic phosphates, chlorides, albumins, C-reactive proteins (hsCRP), total cholesterol (TC), HDL-C, LDL-C, and triglycerides (TG). The immunoassay analyzer ARCHITECT i1000sr  determined the levels of insulin, hs troponin I, TSH, fT3, and fT4 levels. Insulin resistance as HOMA-IR was calculated according to the formula: glucose  at +4 °C and kept there for 1 h at +4 °C. After lysis, slides were gently washed with distilled water and kept for 20 min in the freshly prepared cold denaturation solution at +4 °C , and then placed into a horizontal electrophoresis tank in a new denaturation solution at +4 °C for a further 20 min during electrophoresis at 0.8 V/cm and 300 mA. All of the steps were performed under dim light. After three washes in a neutralization step with 0.4 M Tris-HCl (pH 7.5), for 5 min each, slides were immediately processed further, stained with ethidium bromide at a concentration of 20 μg/mL, and analyzed under the Olympus BX51 (200× magnification) fluorescence microscope using the Comet Assay IV software for image analysis . A total of 100 nucleoids (50 per slide) were measured per sample. The tail intensity parameter  was used. Samples from both gels of the same individuals were compared, and details will be explained in the statistical part.For the alkaline and Fpg comet assays, 2 mL of whole fresh human blood from each volunteer was collected in an EDTA vacutainer, transported to the Institute for Medical Research and Occupational Health at 4 °C, light protected, on the same day, and immediately processed further. The procedure for the alkaline comet assay was described previously , and det2EDTA, and 0.2 mg L−1 BSA, pH 8.0, Sigma-Aldrich, USA) for 5 min each time. Fpg gels were treated with 70 µL Fpg enzyme each , diluted in Buffer F, while controls were treated only with 70 µL Buffer F and covered with coverslips. After 60 minutes of humid chamber slide incubation at 37 °C, coverslips were removed, gels were washed with distilled water, and then followed the procedure as described for alkaline comet assay, but all the equipment, treatments, lysis, denaturations, electrophoresis, and neutralizations, were performed separately from the regular alkaline comet assay to avoid any influence on the results.The blood samples in the same agarose concentration (70 µL of cell/gel mixture) and 2 gel system were also used for the Fpg comet assay. In this assay, we had more slides, since duplicates should be performed for the alkaline comet assay with the treatment with only buffer F and duplicates should also be performed for the Fpg enzyme plus buffer F treatment. Up to the lysis, all the steps were the same as for the regular alkaline comet assay. After lysis, slides were immersed in 3 changes of Buffer F . The descriptor of interest was tail intensity , measured in agarose-embedded cells . Fpg TI Net value was calculated after subtraction from the Fpg control buffer values [Slides were scored under an epifluorescence microscope , under 200× magnification. The level of DNA damage in individual cells was assessed with Comet Assay IVr values . All samr values , the mea−2 [For both the alkaline and Fpg-modified alkaline comet assay, we did not use internal control in this study. In our previous studies, collecting databases on more than 19,000 individuals all over the world, we have determined the reference values for TI values for the healthy population of up to 9%, the cancer-free population of up to 12.4%, cancer-prone/cancer cases 17.7%, and deceased population 18% [−2 ,25.2 flask through the addition of 0.6 mL of heparinized blood under sterile conditions into a prewarmed cell culture medium. The cell culture medium consisted of 6 mL RPMI-1640 medium, 1 mL fetal calf serum (FCS), 0.02 mL phytohemagglutinin-L, and 0.01 mL antibiotic solution (100 IUmL−1 penicillin and 100 mg mL−1 streptomycin) and was prepared freshly and kept at 37 °C for at least 1 h before cultivation. Cultures were successively incubated at 37 °C with 5% CO2 in the air in a humidified atmosphere in the cell incubator . After 44 h of incubation, cytochalasin-B was added to the cultures in sterile conditions at a final concentration of 6 mg mL−1 for cytokinesis blocking. After 72 h of incubation, cells with the medium were transferred into glass centrifugation tubes, which were run at 450× g for 10 min in a centrifuge  using a swing bucket rotor. Then, the solution was removed, and the cell pellet was gently mixed with a cold, mild hypotonic solution (75 mM KCl) and left at room temperature for 10 min. After supernatant removal, the cell pellet was fixed with a fresh mixture of cold methanol/acetic acid (3:1 v/v) . The treatment with the fixative was repeated three times, and the cell pellet, dissolved in a minimal volume of fixative, was seeded on clean, cold microscopic slides , dried, and stained for 10 min with 5% Giemsa (pH 6.8) prepared freshly in distilled water . Microscope analysis was performed at 400× magnification using a light microscope . MNi (a biomarker of chromosome breakage and/or whole chromosome loss), nucleoplasmic bridges , and nuclear buds  were scored in 2000 binucleated lymphocytes with well-preserved cytoplasm per subject. A total of 1000 lymphocytes per donor were scored to evaluate the frequency of cells with 1–4 nuclei  within the same cytoplasm. The cytokinesis-block proliferation index or nuclear division index (NDI) was calculated according to the following formula, with M1–M4 representing the number of cells with 1–4 nuclei, and N the total number of cells scored (1000) [Details of the method can be seen in our previous publication , but we d (1000) .NDI = (The frequency of apoptotic (programmed cell death) and necrotic cells in 1000 lymphocytes per subject was also scored, and the scored DNA damage parameter frequency (per 2000 binucleated lymphocytes (BN)) was calculated and also expressed as 1000 BN.For this study, we did not use an internal control for the assay. In our previous study, we established the reference MN values for a healthy Croatian population based on 200 individuals (up to 12.5 or 13 MN per 1000 BN), which also corresponded to the HUMN group, and according to the HUMN group, reference values for NB (up to 5 per 1000 BN) and NPB (up to 10 per 1000 BN) ,15,16,39® 13.05.0.17 software package  for the analysis, descriptive statistics were carried out (mean ± standard deviation (SD), standard error (SE), and median, range). The EPIC-Norfolk food questionnaire was analyzed with FETA software [p < 0.05. Except for statistical significance, all data with a decimal point were set to two decimals. For DNA damage modeling for TI values in the comet and Fpg comet assays and for MN, NB, and NPB values in the MN cytome assay, we used the free software R statistical programming language . The stepwise regression model was performed to investigate the relationship between different features that can affect measured tail intensity, MN, NB, and NPB and give us a model prediction for the used anthropometric and biochemical parameters. Stars used in these graphs are *—p value < 0.05, **—p value < 0.01, and ***—p value < 0.001.Using the Statisticasoftware . AnthropWe wanted to investigate the relationship between different biomarkers on the group level, then on the gender level, and finally on the age level (with different age categories). Since those categories can possibly influence the results, and establish how dietary inflammatory index and food ingredients are connected to inflammation and influence our results and individual DNA damage results, the results are also grouped in that way. This manuscript is supposed to serve as the basis for future meta-analyses, but since the data are huge and it is confusing to navigate through all the data, we will present the main findings in the main manuscript and the whole tables will be in the Individual demographic details can be seen in In general, we had 36 females and 17 males. There were only 3 smokers; two of them smoked the same number of cigarettes per day (one for 30 and the other for 4 years), while the third person smoked electronic cigarettes. In the entire group, 18 subjects were highly educated (university degrees), and the rest had middle or lower school finished. Seven subjects declared occasional exposure to pesticides , and the other two reported twice-yearly exposure to wood or metal varnish (due to hobbies and activities they do in their free time).Among them, 25 individuals had closer family relatives with tumor/cancer diseases. Eight participants had a tumor surgically removed in the past. Hypertension was diagnosed among 28 individuals, 5 had asthma, 19 were with diabetes type II, 4 with hypothyroidism, and 18 with dyslipidemia. Some of them occasionally used vitamins or supplements, but not regularly. Eighteen of them were physically active .Considering medication, 30 reported the use of antihypertensives, 17 the use of antileptics, and 17 the use of antidiabetics.As for the age of the group, mean, median, SD, SE, and range were: 51.15 years; 51.41; 11.59; 1.59; 26.35–68 years.The results reported in Weight changes correlated with BMI, BMR, BFM, and SMM increases, but there was a very weak, although still significant, correlation between BMR and BMI.Next, we wanted to investigate the trends in the values of biochemical parameters . We shouCBC was, according to the mean and median values, in the range of reference values, although for each parameter there was at least one person who had higher levels than the reference range. Lipid profile , glucose and HOMA-IR, hsCRP, and urates were higher than the reference range of values.Looking at an individual level, 20 individuals had a lower mean corpuscular volume of red blood cells (MCV) values; 16 of them had higher neutrophil counts; 25 had higher glucose levels; 31 had higher urate values; 34 had higher hsCRP values; 34 had higher TC values; 33 had higher LDL-C levels; 32 had higher TG values; 11 had higher insulin levels; and 48 had higher HOMA-IR values than the reference values. In 3 individuals, TSH levels were above the reference range.In From the elevated levels of biochemical parameters, only a few of them correlated with the anthropometric ones. Insulin levels moderately correlated with weight; urate levels moderately correlated with SMM and BMR; and HOMA-IR levels moderately correlated with weight and BMI.TC demonstrated a very strong correlation with LDL-C and moderate with TG, HDL-C, and hs troponin. Hs troponin is a biomarker of myocardial injury and independently associated with obesity and BMI . TherefoAlthough levels of inorganic phosphates and chlorides were within the reference range, we will mention them here since in this table and also in the table for genomic instability, they showed a slightly moderately positive correlation with DNA damage levels, elevated anthropometric parameters, and also hs troponin. Although the correlation is moderate but present in all tables, this is also an interesting finding that should be further explored with a larger number of individuals to get some final answers. Inorganic phosphate has been associated with different chronic diseases, including type 2 diabetes mellitus, obesity, and even cancer, and linked with tissue damage and antiproliferative effects depending on the cell status .cytome assay demonstrated a decrease in nuclear proliferation index (less than 2), with more mononuclear (M1) than binuclear cells (M2). Among the subjects, higher frequencies for apoptotic cells were also noticed. Although the group mean value of micronucleus frequency was in the reference range, there were individuals with higher levels of damage. The same goes for the NB and NPB parameters, again pointing out that there should be further analysis at the individual level.MN Briefly, the results demonstrated that there was a moderately negative correlation between M2, M3, and M4 cell frequency, and NDI with the elevated anthropometric parameters of weight, BMI, and BFM. A positive correlation was seen with M1 cell frequency. This means that the increase in weight, BMI, and BFM in the severely obese is influencing the lowering of the mitotic ratio of the cells counted in the micronucleus assay. Similar was seen for MN mitotic parameters and levels of inorganic phosphates, chlorides, and hs troponin, although the correlation was moderate. Inorganic phosphates, chlorides, and hs troponin, but also glucose levels, had a moderate correlation with the level of necrotic cells. Alkaline comet and micronucleus cytome assays did not show any correlation with other parameters. In the alkaline comet assay, TI had a moderately positive correlation with TG and chlorides, while weak correlations were observed for apoptosis, glucose, and inorganic phosphates.cytome assay values  to −8.87 (strongly anti-inflammatory). The “Food parameter-specific DII scores” are then summed to create the “overall DII score” for an individual and for the group. For the entire group, the DII mean, median, minimum, maximum, and standard deviation values were: 2.06, 2.18, −3.37, 6.73, 2.39, and 0.33. DII demonstrated a weak positive correlation with biochemical parameters: leukocytes (0.31), neutrophils (0.27), lymphocytes (0.27), monocytes (0.29), and a negative correlation with hsCRP values (−0.29). DII correlated with micronucleus Daily consumption in grams for 130 food ingredients in the last year from the FFQ questionnaire on the group levels is presented in On a daily basis, in the entire group, there were 23 consumers of white bread and rolls , 13 of whole meal bread and rolls , and 12 of brown bread and rolls (4 once and 8 twice or thrice daily). There were 34 daily coffee with caffeine consumers  and 16 tea consumers  and among them, there were 12 who were daily adding sugar to coffee/tea. Ten eat chocolate regularly daily (4 once and 6 twice or thrice). Seven eat cheese (5 once and 2 twice or thrice daily). Of course, there were 13 regularly consuming garlic daily , 15 onions once per day, 13 consumed apples daily (9 once and 5 twice or thrice), 7 oranges (6 once per day and one twice or thrice) and 7 consumed once per day a banana.On a weekly basis, there were 25 consuming beef , 31 pork consumers , 43 chicken , 25 bacon  and 24 ham weekly consumers . Considering potato consumption, there were 38 boiled potato consumers per week , 21 pommes frites , 33 roast potato consumers , and 22 potato salad consumers (19 once and three 2–4 times per week). There were 35 weekly egg consumers , 30 consumed cottage cheese , and 26 cheese weekly . Among them, there were 29 weekly white rice consumers  and 32 white pasta consumers . Thirty-three consumed meat soup weekly , 16 ice cream , 18 chocolate , 10 chocolate bars , and 14 ready-made buns . There were also 25 consumers of low-fat yoghurt  and 14 full-fat yoghurt consumers .Results reported in the tables demonstrated that older individuals lower their energy intake, sugar intake, and alcoholic beverage consumption. Proliferation parameters and less damage in the comet assay correlated with vegetable intake. Damage levels measured by the comet assay and the Net Fpg comet were negatively correlated with cereals and cereal products and preserves and snacks. Micronucleus frequency was also correlated with the consumption of eggs and egg dishes. The inflammatory dietary index was negatively correlated with the intake of cereals and cereal products, fish and fish products, fruit, and milk. TG negatively correlated with consumption of the healthy fats and oils. Vitamin C negatively correlated with the Net Fpg comet assay values and proliferation parameters recorded by the micronucleus assay.The dietary inflammatory index for the entire group did not demonstrate high DII at the group level, which was a good fact since it is highly recommended that DII be as low as possible. However, when individual DII was calculated , there wWhen divided into two groups by sex , the intp = 0.04, more in the female group). However, males used more alcoholic beverages, and females used more cereals, milk products, healthy oils, fruits, vegetables, preserves, and snacks. When we analyze the differences in the consumption of certain food groups by sex, only the intake of nuts and seeds was significantly different , micronucleus, or comet assay values, but the proliferation index was higher in the female group. The female group had generally lower damage measured with micronucleus and comet assays, except for the oxidative damage (Fpg), which was higher in that group.After the subjects were divided into different age groups, we found some significant values . We deciWhen divided into groups with ages ≥60 and <60 , subjectThe group of subjects younger than 60 was further divided into subgroups of 51–59 years, 41–50 years, and <40 years , and theComparison of the ≥60 group with the 41–50 group demonstrated that the older subjects had highly significant values for BMI and hs troponin I. The 41–50 group had significantly higher values for Ca (mg), chlorides, iron (mg), energy kJ, carbohydrate maltose (g), sodium, phosphorous, carbohydrate sucrose, vit B1-thiamin, zinc, total fat, MUFA, SFA, alcoholic beverages, meat and meat products, sugars, and TG.Comparison of the group ≥60 with ≤40 demonstrated that the older subjects had significantly higher values for carbohydrate maltose, SFA, hs troponin I, folate, fT4, HOMA-IR, M1, M3, M4, NDI, and MN. Group <40 had significantly higher levels of sugars.Comparison of the group 41–50 with ≤40 demonstrated that the older subjects had significantly higher levels of niacin, proteins (g), vit B2-riboflavin (mg), vitB1, nitrogen (g), vit B12, zinc (mg), meat and meat products, and MN.Comparison of the group 51–59 with ≤40 demonstrated significantly that the older subjects had higher levels for hs troponin I, TSH, BMR, M1, and MN. Group <40 had significantly higher levels of sugars (g), fT3, HOMA-IR, M2, M3, M4, and NDI.A comparison of the 51–59 group with the 41–50 group demonstrated that the younger subjects had significantly higher levels of hs troponin I and M3.When all three subgroups  were inter-compared , the 41–To additionally prove the age effects on the parameters of interest, we further divided the entire group into age groups, >50 (n = 27) and ≤50 years (n = 26)  . ObtaineConsidering that by conducting all analyses, which are documented in the above-reported tables and As for oxidative DNA damage, there are no referent values, and the values were supposed to be around 0%, but on the group level the average was 6%, but we had 20 individuals exceeding those values. As for the comet assay, the existing literature from our group ,18 estabIn Next, we wanted to check what was common in individuals with higher DNA damage see  when theConsidering that we had 8 individuals with former tumors and 25 individuals with a family history of tumors, we wanted to explore the level of DNA damage in both assays for those individuals . The nonHowever, when the individuals with tumor history were checked, we noticed that most of them had higher values for TI  with most of them being in the cancer-prone group for the comet assay (five of eight) . For thecytome assay. In all of our models, the presence of previous tumors or a family history of tumors did not influence the model.At last, we made multiple regression models connecting the DNA damage values found in all three assays with the analyzed biochemical and anthropometric parameters. These relationships were already explained in previous analyses; this is only a graphical presentation of the different positive and negative influences on the DNA damage predictions. These predictions will help in new investigations on a higher number of patients and also serve as the model which will be either confirmed or changed with the higher number of participants. The novelty of this study is that, although performed on a relatively small group of individuals (n = 53), for the first time, three different DNA damage assays  were used in the same study on the same whole blood samples of severely obese individuals. The study demonstrated that all anthropometric parameters were elevated in the group and that they all correlated together, although BMI and BMR showed weak correlations, demonstrating again the need to use both of those parameters in assessing the severity of obesity with anthropometric measurements.Although the anthropometric measurements did not strongly correlate with biochemical parameters in general, we were able to show a moderate correlation of insulin, HOMA-IR, and urate levels with weight, SMM, BMR, and BMI. This only demonstrated that both types of approaches, anthropometric and biochemical ones, are much needed in obesity and its severity assessment.In terms of biochemical parameters, the lipid profile , glucose, HOMA-IR, hsCRP, and urates were found to be higher than the reference range of values among the majority of the participants in the group, together with a lower MCV and a higher neutrophil count among the other half of the group. Inorganic phosphates and hs troponin levels, although in reference range levels, in all three types of the parameters  demonstrated some influence (correlations).Even though the FFQ frequency did not demonstrate that some types of consumed food will have an impact or clear influence on any of the anthropometric, biochemical, or elevated DNA damage parameters, the trend of eating some types of food daily, weekly, and monthly was noticed in groups with higher DNA damage than expected .DII itself demonstrated a weak correlation with oxidative DNA damage measured using the Fpg comet assay, and in our modeling of TI prediction, DII, together with lipid profile parameters, urates, inorganic phosphates, MCV, and even waist-to-hip ratio, was among the parameters most strongly influencing the prediction of DNA damage in severely obese individuals. It was also seen that DII values were the highest in the ≤40 years age group. We demonstrated that increases in BMR, BFM, and BMI are connected with a decrease in the proliferation index, an increase in the frequency of mononuclear cells, and an increase in the DNA damage parameters in all three assays. Furthermore, inorganic phosphates and hs troponin levels, although in reference range levels, again were demonstrated to have some influence (correlations). BMI, BMR, BFM, SMM, and weight were the highest in the 51–59 years age group. This sub-group had the highest level of damage in terms of micronucleus frequency, apoptosis, and comet assay values. The highest NB and NPB frequencies were found in the 41–50 years age group.DNA damage assays, especially the comet assay, demonstrated sensitivity in detecting individuals with higher genomic instability and, therefore, an increased risk of further developing obese comorbidities, such as cancer. Among the participants with previous tumors, or a family history of cancer, who did not show different results from other participants that were less suspected to have genomic instability, the assay demonstrated that it could track at least half of them in the cancer-prone group with the higher DNA damage, We also demonstrated that all three assays should be used together in the assessment of the severity of obesity and its comorbidities. Each assay demonstrated different types of damage that occur at different points in the life spans of severely obese individuals. In the future, both DNA damage and biochemical parameters should be combined with anthropometric ones for further monitoring of obesity. Since the study group consisted of a small number of individuals and the correlation was found to be weak or moderate, the experiment should be replicated, and the experimental design should include a larger number of participants in order to confirm our results and possibly provide a stronger correlation. Such a design could also result in weak correlations, demonstrating the need to use the previously mentioned three different parameters  in the assessment of obesity severity and revealing the DNA damage-prone individuals.Although still scarce, knowledge on disease-causing obesity biomarkers holds promise to be used for a more refined diagnosis of obesity beyond anthropometric measurements, for more precise identification of persons at high risk of obesity/disease/genomic instability/cancer development, and in individual systems medicine/therapy. Combining biomarkers should also give us a better insight into the disease development and treatment approach.−2), BMI is still a good predictor of obesity and adipose-associated risks [There are reports characterizing BMI as not an accurate marker of obesity ,47,48,49ed risks ,47,48,49ed risks , and inded risks , and coued risks . With thThe major cause of severe obesity is a long-term imbalance between energy intake and energy expenditure, which leads to weight and BMI gain and consequently an increase in all other anthropometric parameters ,54. Suchcytome assays are sensitive and appropriate methods for DNA damage assessment but have not yet been fully explored in their potential for clinical use with other assessment biomarkers [At the same time, BMI and BMR were connected with a decrease in cell proliferation and DNA damage creation. We already mentioned in the introduction part that obesity is associated with an elevated risk of several NCDs, with evidence that DNA damage, mostly oxidative but also DNA damage in general, plays a key role in the development of most common NCDs, including cancer (even 13 different types), and that among other assays, comet, Fpg comet, and micronucleus omarkers ,13,19. Oomarkers ,58. Inteomarkers , but an omarkers . With thomarkers . Also, oomarkers ,19. The Although the comet assay, after an analysis of a huge registry/database of more than 19,000 individuals, has established certain values for healthy, cancer-free, cancer-prone, and deceased populations  ,19, the From biochemical parameters, elevated levels of glucose demonstrated a connection with an increase in apoptotic and necrotic cells and cells with higher damage in the comet assay. Hs CRP by itself did not demonstrate a connection with DII or DNA damage, but it was found that a decrease in MCV was connected with elevated HOMA-IR and insulin levels, and platelet concentrations were connected with hsCRP. The results demonstrated an association between DNA damage levels and cell proliferation with anthropometric measurements and severity of obese status, together with inorganic phosphates, hs troponin I levels , and elevated levels of urates, insulin, and HOMA-IR. An increase in calcium levels, inorganic phosphates, and chlorides demonstrated a connection with comet assay DNA damage, necrosis, a decrease in cell proliferation, and an increase in MN frequency.−2) is connected with a significant reduction in the disease-free years  of 7 to 8 (up to 10) years in both males and females, smokers and non-smokers, physically active, and inactive, and across the social hierarchy, in people between 40 and 75 years of age who did not have any disease when first entering the study [Severe obesity is associated with glucose intolerance, hyperinsulinemia, insulin resistance, hypertriglyceridemia, decreased high-density lipoprotein (HDL) cholesterol levels, and high blood pressure and can be a precursor of cardiovascular disease and type 2 diabetes mellitus (T2DM) . Changes 8 up to  years inFurther steps should include study replication with a larger number of participants, the development of a registry of obese patients, which would simplify the monitoring and follow-up processes, and connecting that registry with the mortality registry. In addition, individuals should be educated on how to eat healthy and given appropriate diets, explanations about the type of food with proinflammatory or anti-inflammatory potential, and how to use that information. All of these suggestions could be performed under medical surveillance and should also include BER and NER comet assays in order to check whether DNA damage has been reduced after the diets and whether repair mechanisms have gained their activity again. All significant biomarkers found in this study should be regularly used in the systemic and individual approaches to each obese and especially severely obese patient. New data on those biomarkers would also give the possibility for new data meta-analysis and provide new guidelines for future treatment and prevention of severe obesity and cancer, and maybe obesity in general.


KCNK4 gene, predominantly distributed in neurons, plays an essential role in controlling the resting membrane potential and regulating cellular excitability. Previously, only two variants were identified to be associated with human disease, facial dysmorphism, hypertrichosis, epilepsy, intellectual/developmental delay, and gingival overgrowth (FHEIG) syndrome. In this study, we performed trio-based whole exon sequencing (WES) in a cohort of patients with epilepsy. Two de novo likely pathogenic variants were identified in two unrelated cases with heterogeneous phenotypes, including one with Rolandic epilepsy and one with the FHEIG syndrome. The two variants were predicted to be damaged by the majority of in silico algorithms. These variants showed no allele frequencies in controls and presented statistically higher frequencies in the case cohort than that in controls. The FHEIG syndrome-related variants were all located in the region with vital functions in stabilizing the conductive conformation, while the Rolandic epilepsy-related variant was distributed in the area with less impact on the conductive conformation. This study expanded the genetic and phenotypic spectrum of KCNK4. Phenotypic variations of KCNK4 are potentially associated with the molecular sub-regional effects. Carbamazepine/oxcarbazepine and valproate may be effective antiepileptic drugs for patients with KCNK4 variants.The  It encodes the potassium two pore domain channels subfamily K member 4 (K2P4.1), a lipid-and mechano-sensitive K+ ion channel with 393 amino acids. The K2P4.1 channel, mainly distributed in the membrane of neurons, plays an essential role in controlling the resting membrane potential and regulating cellular excitability   . It is and KCNH1 . With inepilepsy . There rKCNK4 variants were identified in two unrelated cases, including one with Rolandic epilepsy and one with the FHEIG syndrome. The molecular structure alteration was analyzed by 3D protein modeling. We further reviewed all KCNK4 variants and analyzed the molecular sub-regional effects. The present study expanded the phenotypic and genetic spectrum of KCNK4.In this study, we performed trio-based WES in a cohort of patients with epilepsy without acquired causes. Two novel likely pathogenic 2.2.1.A total of 156 children with epilepsy without any acquired causes were recruited from Guangdong 999 Brain Hospital from June 2020 to March 2021. Clinical information of the affected individuals was collected, including age at onset, type, and frequency of seizures, family history, systemic and neurological findings, and effective antiepileptic drugs. The structural abnormalities were detected by brain magnetic resonance imaging (MRI) scans. Long-term video electroencephalography (EEG) monitoring records were performed with electrodes being arranged according to the international standard of the 10–20 reduced montage system. The EEG results were reviewed by at least two qualified electroencephalographers.This study adhered to the principles of the International Committee of Medical Journal Editors concerning patient consent for research or participation and received approval from the ethics committee of the Guangdong 999 Brain Hospital. Written informed consent was provided by the patient’s legal guardians.2.2.Blood samples of the probands and their parents were collected for identifying the source of variants. Genomic DNAs were extracted from blood samples using the Qiagen Flexi Gene DNA kit . WES was performed using a NextSeq500 sequencing instrument  following previously described standard procedures . The seq2.3.in silico tools, including CADD, GERP, SIFT, Fathmm-MKL, fitCons, LRT, M_CAP, polyphen2_HDIV, PolyPhen2_HVAR, MutationTaster, MetaSVM, MetaLR, GenoCanyon, VEST3, Eigen, ClinPred, REVEL, phastCons, phyloP, SiPhy, and PROVEAN. The scores of these tools were obtained from the VarCards database  was used for data processing. The two-sided Fisher exact test was used in the aggregate frequency analysis, which was recommended by ClinGen to compare the frequencies of the controls . p < 0.03.3.1.KCNK4 gene were identified in two unrelated patients, including one with FHEIG syndrome and one with Rolandic epilepsy  in the epilepsy ; Table 1KCNK4 variants identified in this cohort was statistically higher than that in controls, including the gnomAD-all populations , the gnomAD-East Asian , the ExAC Population , the ESP 6500 Population , the 1000 Genomes Population , and the Epi25 WES Brower Population .The two variant was not presented in any controls, including the population in the gnomAD database, the ExAC database, the 1000 genome database, and the Epi25 WES Brown database . The aggKCNK4 variants identified in this study were predicted to be “Damaged” by numerous in silico tools,  , absence in controls (PM2), and being predicted to be damaged by multiple in silico tools  . The two species . Accordiols PP3; .No pathogenic or likely pathogenic variants in other epilepsy-related genes were identified in the two patients .3.2.The detailed clinical information of the patients were summarized in The boy with the p.Pro233Leu variant presented a mild phenotype of Rolandic epilepsy. He suffered from FBTCS 1–3 times per year since 1.5-year-old. Seizure-free was achieved after being treated with valproate (21 mg/kg/day) and oxcarbazepine (9.1 mg/kg/day). He presented a purely epileptic phenotype without any other phenotype. EEG showed epileptic discharge in the Rolandic area with right-side dominance .The two patients were born to unrelated parents after an uneventful pregnancy. The brain MRI detected no abnormalities.3.3.KCNK4 gene encodes a highly conservated lipid-and mechano-sensitive K+ ion channel with 393 amino acids. It contains four transmembrane segments (TM 1–4) and two pore domains . Nevertheless, the recurrence of the FHEIG syndrome in Case 2 provided further clinical evidence in supporting the association between KCNK4 and the FHEIG syndrome. The mechanisms of multisystem symptoms of KCNK4 are of great challenge and warrant further studies.Previously, two syndrome . Clinicasyndrome . The KCNKCNA2, KCNT1, KCNT2, and KCNB1; KCNN3 and KCNH1)  . With inPrevious studies suggested that the phenotypic variation was potentially associated with the location of variants, which was known as the molecular sub-regional effect . FunctioKCNK4 variants (including two from this study) were identified in six patients to be associated with human disease .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.


The mental health of the elderly is vulnerable during public health emergencies, such as the COVID-19 pandemic, and the risks of depression is increased. The study aimed to explore the effects of physical health, the Big Five personality traits, media use, and different kinds of perceived social support as predictors of levels of depression among the elderly during the COVID-19 pandemic. A cross-sectional survey was conducted in more than 120 cities in China with a sample of 1147 older adults, and hierarchical regression was established to examine the predictors of the level of depression. The results showed that almost half of the elderly (48%) had a status of mild or much more severe depression during the COVID-19 pandemic. The level of depression was negatively associated with physical health, extraversion, conscientiousness, agreeableness, and family support, while it was positively associated with neuroticism and media use. The study emphasized the effects of physical health, the Big Five personality traits, media use, and perceived social support from family as the predictors of levels of depression, providing further measures to help the elderly better react to a public health emergency. During public health emergencies, such as the coronavirus disease 2019 (COVID-19) pandemic, the risk of psychological problems among the public, such as depression, increases . This siIt is accepted that mental health has a close connection with physical health . Chen anThe general patterns of individual differences, such as personality, may have an impact on how individuals respond to a public health emergency , and theEach specific Big Five personality trait showed different correlations with health outcomes. Extraversion was proven to be negatively associated with psychological problems and somatic health symptoms . ConscieFor depression, these five kinds of personality traits might have distinctive influences, and they might cause different effects on different genders . CarreirMedia use is an important component of modern daily life, and it is inherently personal. When focusing on how individuals use each kind of media, personal characteristics are crucial observational factors. It has been proven that the Big Five personality traits were associated with certain media use behaviors. For example, total Internet usage had negative associations with agreeableness, conscientiousness, and extraversion , while tMedia use can act imperceptibly on individuals’ mental health. During the COVID-19 pandemic, the mass media have played an important role in conveying health-related information, accessing perceived social support, and so on . HoweverIn contrast with other age groups, the limitations of the physical and cognitive abilities of the elderly lead to them using smartphones less and digital media differently ,27. ThesPerceived social support refers to the cognition and appraisal of individuals’ reliable connections with others , which iPerceived social support has always been regarded as an important positive factor in both physical health and mental health ,35, helpAgainst the background of the COVID-19 pandemic, the impact of perceived social support as a protective factor of mental health has attracted much attention. Preventive measures, such as home quarantine and maintenance of social distance, might reduce perceived social support, especially for the elderly , which iBased on the above discussion, this study examines the predictive power of physical health, Big Five personality, media use, and different sources of perceived social support on depression among the elderly during the COVID-19 pandemic. The research questions and hypotheses were formulated as follows. RQ1: Was physical health significantly related to the level of depression among the elderly during the COVID-19 pandemic?RQ2: Were the Big Five personality traits significantly related to the level of depression among the elderly during the COVID-19 pandemic?H1: Extraversion is negatively associated with the level of depression.H2: Conscientiousness is negatively associated with the level of depression.H3: Agreeableness is negatively associated with the level of depression.H4: Neuroticism is positively associated with the level of depression.H5: Openness is negatively associated with predicting the level of depression.RQ3: Was overall media use significantly related to the level of depression among the elderly during the COVID-19 pandemic?RQ4: Was perceived social support significantly related to the level of depression among the elderly during the COVID-19 pandemic?H6: Family support is negatively associated with the level of depression.H7: Friends’ support is negatively associated with the level of depression.H8: Significant others’ support is negatively associated with the level of depression.The demographic factors that might be correlated with the level of depression ,41,42, sA cross-sectional survey using multistage sampling was conducted in China from 10 July to 15 September in 2021, which was during the COVID-19 pandemic. According to the Seventh National Population Census of the People’s Republic of China in 2021, quota sampling  of the selected elderly residents from 120 cities was finished. The participants had to meet the following criteria: (1) aged 60 years old or older; (2) Chinese nationality (from the People’s Republic of China); (3) willingness to participate in the study voluntarily; (4) ability to complete the network questionnaire survey on their own or with the help of the investigators; and (5) ability to understand the meaning of each question in the questionnaire. A total of 1147 completed questionnaires were valid for the study. The questionnaire consisted of two parts. The first part was the demographic characteristics , and the second part included scales that were utilized to measure depression, physical health, personality, media use, and perceived social support. To measure the level of depression symptoms among the elderly, the Patient Health Questionnaire (PHQ-9) was employed, as it is effective for the evaluation of depression disorder . The PHQThe EQ-5D-5L is a generic instrument to assess individual health, which includes dimensions of mobility, self-care, usual activities, pain or discomfort, and anxiety . In the A 10-item short version of the Big Five Inventory (BFI-10) was employed to measure the main personality traits of the elderly, including five dimensions: openness, conscientiousness, extraversion, agreeableness, and neuroticism. This was a reliably abbreviated version of the BFI-44. The BFI-10 used a 5-point Likert scale ranging from 1  to 5  . The scoMedia use was measured using the following question: How often do you use  in daily life each week? (1) never use (=1); (2) ≤1 day/week (=2); (3) 2–3 days/week (=3); (4) 4–5 days/week (=4); (5) 6–7 days/week (=5). Cronbach’s α of media use was 0.753 in this study. The scores for each kind of media were added to measure overall media use. The total score ranged from 7 to 35, and higher scores indicated more media use. The descriptive statistics of the use frequency of different kinds of media are presented in The Multidimensional Scale of Perceived Social Support (MSPSS) was applied to “assess perceptions of social support adequacy from three specific sources [to] measure the basic figures of individual personality” . The MSPThe study utilized SPSS 25.0 to conduct the statistical analysis. First, descriptive statistics were conducted to examine the demographic characteristics of the participants in the current study. Correlation analysis and hierarchical regression analyses were then utilized to analyze the relationship between depression and the Big Five personality traits, media exposure, and different sources of perceived social support.In total, 1147 participants were recruited for the present study , includiThe median scores and inter-quartile range (IQR) of specific scales were as follows: PHQ-9, 4.00 (1.00–8.00); EQ-4D-5L , 19.00 (18.00–20.00); openness, 6.00 (5.00–7.00); conscientiousness, 7.00 (6.00–8.00); extraversion, 6.00 (5.00–7.00); agreeableness, 7.00 (6.00–8.00); neuroticism, 6.00 (5.00–6.00); media use, 17.00 (13.00–21.00); significant other’s support, 20.00 (17.00–24.00); family support, 21.00 (17.00–24.00); and friends’ support, 20.00 (16.00–24.00). p < 0.001), conscientiousness , extraversion , agreeableness , significant other’s support , family support  and friend’s support . In addition, depression had a significantly positive association with neuroticism  and media use . There was no significant association between openness and depression . The Spearman correlation coefficient was utilized to examine the correlation between related factors, and the results are shown in 2Adjusted = 0.148, p < 0.001). The Big Five personality traits  were added in the second model (step 2) . Media use was then added in step 3 , and three dimensions of perceived social support  were added in the final model (step 4) . The residual error was approximately normally distributed. Hierarchical regression was conducted to examine the predictors of depression among the elderly during the public health emergency . The regp > 0.05), age , per capita monthly household income  and marital status  were not predictors of depression. Physical health could negatively predict the level of depression . For the Big Five personality traits, conscientiousness , extraversion  and agreeableness  were the negative predictors of depression, neuroticism  was a positive predictor of depression, and openness  could not predict the level of depression. Media use  could positively predict the level of depression. For different sources of perceived social support, family support  was a negative predictor of depression, while friends’ support  and significant other’s support  were not statistically significant indicators of depression. In the final model (step 4), gender  had a status of mild or much more severe depression during the COVID-19 pandemic period. WHO guidelines on physical activity and sedentary behavior provide authoritative reference content in this matter [Physical health still showed a protective role in the development of depression. It indicated the importance of maintaining physical health. It is known that physical health has a negative correlation with age, and the elderly have lower levels of physical health . Engagins matter . In terms of the Big Five personality traits, conscientiousness, extraversion, agreeableness, and neuroticism were predictive factors of depression in the elderly. On the one hand, conscientiousness, extraversion, and agreeableness showed significant associations with the level of depression. This was different from the results of Koorevaar’s research, in which agreeableness did not show a significant association with depression . This miMedia use also had an effect on depression among the elderly during the COVID-19 pandemic period. Previous research focused on COVID-19-related media exposure indicated a negative association between media use and mental health . The curIt was interesting that not all sources of perceived social support could protect the elderly from depression. The results indicated that only family support demonstrated a significant inverse association with depression. Compared to youth and younger adults, the elderly perceived lower support . PerceivThis study emphasized the significance of paying attention to depression in the elderly under the background of health emergencies. To take further measures to help the elderly better react to possible depression during a public health emergency, especially a normalized epidemic, focusing on the characteristics and habits of the elderly could provide some coping strategies. The present study conducted a hierarchical regression model to examine the predictive power of physical health, the Big Five personality traits, media use, and three different kinds of perceived social support for the level of depression among the elderly during a public emergency. The results showed that depression was negatively associated with physical health, extraversion, conscientiousness, agreeableness, and family support, while it was positively associated with neuroticism and media use. The study also provided some coping insights for protecting the elderly from possible depression during a public health emergency, especially in the current normalized epidemic period. To protect the elderly from depression or lower the level of their depression during public health emergencies, adopting different guidance based on different personality traits of the elderly can provide advanced monitoring. Second, engaging in proper physical activity or physical examinations on time to maintain physical health can strengthen the physical basis for preventing psychological problems. In addition, focusing on media use and guiding the elderly to use media properly or learn beneficial functions are all necessary. Moreover, families should take better care of the elderly in the family, as perceived social support from the family is a forceful power to protect them from depression during public health emergencies. In the study, the 10-item short version of the Big Five Inventory (BFI-10) was utilized to measure personality traits among the elderly, but further studies could utilize the scale containing more items to measure the Big Five personality traits, such as the 44 items of the Big Five Inventory (BFI-44), or try to examine the association between more multidimensional personality traits and depression. In addition, the correlation between further media use, such as the preferences for media content and depression is valuable to explore.


The most common HNC sites were the nasopharynx (25.3%), hypopharynx (22.8%), and oral cavity (22.2%). Carcinoma was the most common diagnosis (87.6%), followed by lymphoma (5.6%). Most patients’ tumors were at a locally advanced (22%) or metastatic stage (47%) of HNCs at the time of presentation. Further studies to identify risk factors for HNCs, particularly for the most prevalent types in central Sudan, are needed. In addition, Sudan requires capacity building for cancer, including a national cancer registry.Head and neck cancers (HNCs) are prevalent in Sudan, but the reasons for this and the incidence of different types of HNCs are not well understood. A cross-sectional retrospective study was conducted to provide baseline data on the epidemiology of HNCs among patients treated at the National Cancer Institute (NCI) in central Sudan. All cancer cases from 2016 to 2020 were retrieved from the NCI records. Of the 9475 new cancer patients who were registered at the NCI during the study period, 1033 (11%) had HNCs, of whom 767 (74.2%) were adults. The mean age of the adult patients was 54.5 years (standard deviation 15.8) and 449 (58.5%) patients were male. The annual incidence in adults was 4/10 Head and neck cancer (HNC) refers to diverse malignancies that affect various anatomical sites in the head and neck area, including the nasopharynx, hypopharynx, oral cavity, larynx, oropharynx, major salivary glands, nasal cavity, and paranasal sinus . Over 90HNC is the seventh most frequent cancer in the world and causes significant morbidity and mortality, with approximately 900,000 new cases and half a million deaths reported annually . In sub-A cross-sectional, retrospective health-facility-based study was conducted. Data were sourced from patient files, pathology records, and radiology reports for new patients who were treated at the NCI from September 2016 to September 2020. The NCI, which is located in the capital city of Gezira, Wad Medani, serves approximately 4 million residents of Gezira and neighboring states, including Sennar, Gadarif, Blue Nile, and Kassala. The retrieved data included age at diagnosis, sex, tumor site, and morphologic or histopathologic tumor information. Tumor topographies were classified according to the 10th version of the International Classification of Diseases (ICD-10) for Oncology. Reports from children; non-Sudanese individuals; and those with dubious diagnoses, benign tumors, skin cancer, cervical esophageal cancer, and central nervous system or eye tumors were excluded.Ethical approval for this study was obtained from the Sudan Medical Specialization Board ethics committee.p ≤ 0.05. IBM SPSS Statistics for Windows V.25.0.  was used to conduct the analyses.Descriptive statistics were used to present the characteristics of the study participants. For continuous variables, the ages of all participants and the ages of each anatomical site group were presented as means and standard deviations (SD). Categorical variables, such as characteristics of participants , included and excluded subjects, distribution of sites of HNCs, and distribution of clinical characteristics by histological types of HNCs, were described using frequencies and percentages . Pearson’s chi-square test was used to examine the relationship between tobacco usage and the anatomical sites of HNCs, as well as gender. Statistical significance was set at 5 “{[1033/(4 × 106 × 5)] × 105}” in the total population and approximately 4/105 “{[767/(4 × 105 × 5)] × 105}” in the adult population.During the study period from September 2016 to September 2020, 89.1% of the 9475 new cancer cases registered by the NCI were excluded because they were not HNCs (8442 out of 9475). Out of 1033 (10.9%) individuals with HNCs as their primary diagnosis, 212 (20.5%) were eliminated due to their age, 41 (4%) were omitted due to their uncertain diagnosis, and 13 (1.3%) were excluded due to their non-Sudanese ethnicity, leaving 767 (74.2%) eligible adults for the study . The estp < 0.001. Further analysis reveals that the prevalence of tobacco usage was higher among males  than females , p < 0.001 , Northern (7/105 population), and Southern Africa (10/105 population) [5 population) and those of other continents, including Asia, Europe, the Americas, and Oceania [Malignancies of the head and neck region are among the most common malignancies in Sudan . Howeverectively . In contulation) . Further Oceania . These v Oceania . This mitoombak users [Toombak is a smokeless tobacco species with high nicotine content, and evidence from Sudan suggests that it is a risk factor for cancer in the oral cavity and perhaps the esophagus [toombak, is stigmatized; therefore, women’s and adolescents’ tobacco usage may be underreported in this study. Furthermore, though literature from Sudan has reported that the prevalence of tobacco use in adults might reach over 45% [toombak, particularly among women and young people.In Sudan, men make up the vast majority of ak users . Toombaksophagus . This stover 45% , tobaccoThe frequency of HNCs at different anatomical sites varies across countries and regions . This stThe current study revealed that the majority of HNCs in central Sudan were epithelial in origin, particularly SCC. This dominance of SCC is well documented in the medical literature ,28,29. LMost of the patients in our study presented with locally advanced tumors (stage III) or metastases (stage IV). Late presentation of advanced HNCs was a common feature in most reports from sub-Saharan Africa ,32. SignThe current study had several limitations, including its retrospective nature and dependence on medical records. Furthermore, the study was limited to a single site; therefore, our findings on HNC cannot be generalized to Sudan as a whole. Nevertheless, the NCI is the sole referral oncology center in central Sudan; hence, our data is a good indicator of the HNC status in this region and sheds light on its characteristics. Sudan lacks a population-based cancer registry and adequate cancer patient coverage ; hence, The incidence rate of HNCs in central Sudan was consistent with that reported in Western and Middle Africa. The most common HNC sites were the nasopharynx, hypopharynx, and oral cavity, with carcinoma being the predominant histological type. Most patients had locally advanced or metastatic HNCs at the time of presentation. Reducing obstacles to early diagnosis and access to appropriate treatment will aid in reducing morbidity and mortality from HNCs. Further studies are needed to identify risk factors for HNCs, particularly for the most prevalent types in central Sudan. Sudan requires capacity building for cancer, including a population-based national cancer registry.


The quantitative and functional analyses of cells are important for cell-based therapies. In this study, to establish the quantitative cell analysis method, we propose an impedance measurement method supported by dielectrophoretic cell accumulation. An impedance measurement and dielectrophoresis device was constructed using opposing comb-shaped electrodes. Using dielectrophoresis, cells were accumulated to form chain-like aggregates on the electrodes to improve the measurement sensitivity of the electrical impedance device. To validate the proposed method, the electrical impedance and capacitance of primary and de-differentiated chondrocytes were measured. As a result, the impedance of the chondrocytes decreased with an increase in the passage number, whereas the capacitance increased. Therefore, the impedance measurement method proposed in this study has the potential to identify chondrocyte phenotypes. The quantitative and functional analyses of living cells and tissues are important for the clinical application of cell therapies such as regenerative medicine. Although conventional analysis methods, including polymerase chain reaction, immunohistology, and flow cytometry using fluorescent dyes, are superior for evaluating cell types and phenotypes in detail, they require complex procedures and expensive instruments. Therefore, a simple, cost-effective, and quantitative evaluation method for cells and biological tissues is required.Electrical impedance measurement is a promising approach for characterizing cell types, cell functions, and biological tissues. It has been used to determine simple cell properties, such as size  and concIn this study, we hypothesized that the accumulation of cells on the electrodes to remove the buffer would improve the sensitivity of impedance measurements, even at lower cell concentrations in cell suspensions. Dielectrophoresis (DEP) is a promising approach for manipulating, patterning, and accumulating living cells ,11,12,13The purpose of this study is to establish an efficient impedance measurement method and improve its sensitivity by dielectrophoretic cell accumulation. We constructed transparent opposing comb-shaped electrodes on glass slides to enable both the DEP and impedance measurements of living cells. Using our proposed method, cells were accumulated on the electrodes by a positive DEP force, and electric impedance measurements were performed. Furthermore, the impedances of differentiated and de-differentiated chondrocytes were measured to verify the effectiveness of our method in identifying the differentiation and de-differentiation of chondrocytes. The efficient evaluation of the electrical impedance of living cells in cell suspensions requires a reduction in the effect of the surrounding buffer. In this study, positive DEP was used to form pearl-chain-like cell clusters . In this3 pieces. The minced tissues were then gently agitated in 0.2% collagenase type II digested with Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12) supplemented with 5% fetal bovine serum (FBS) and antibiotics–antimycotics for 12–16 h at 37 °C. The tissue-digested solution was filtered through a 70-μm nylon mesh filter  to remove the debris. Afterward, the chondrocytes were isolated from the cell-containing solution by centrifugation at 1500 rpm for 5 min and resuspended in phosphate-buffered saline. This procedure was repeated twice to wash the isolated chondrocytes. Finally, after centrifugation at 1500 rpm for 5 min, the chondrocytes were resuspended in a fresh culture medium (DMEM/F12 supplemented with 10% FBS and antibiotics–antimycotics) for cell culture. The cells were cultured on cell culture flasks to reach 80% confluence. After reaching the confluence, the cells were treated with 0.05% trypsin/EDTA and passaged in new flasks. The chondrocytes were cultured for multiple passages to induce de-differentiation. Based on PCR and immunohistology, previous studies reported that multiple passage induced de-differentiation of articular chondrocytes [2, and 59-mM D-glucose in sucrose solution) [Primary and passaged calf articular chondrocytes were used for this study based on previous reports ,25,26,27olution) .In this study, an impedance measurement device supported by DEP cell accumulation was developed. Both the impedance measurements and DEP were performed on opposing comb-shaped electrodes fabricated on a glass slide  a. The elFor DEP, an AC voltage was imposed by a function generator , and an amplifier  was connected to the electrodes b. The apRLCB and electrical components of the cells. Meanwhile, the electrical components of the cells are frequency dependent, consisting of the plasma membrane resistance Rm and capacitance Cm connected in parallel to each other and in series with the cytoplasm resistance Rcyto. Additionally, the impedance of Cm and Rm in parallel is frequency dependent. Under ideal conditions, the cytomembrane capacitance dominates the impedance measurement and prevents electric current from penetrating the cytoplasm at lower frequencies. At higher frequencies, the plasma membrane acts as a high-pass filter for current to penetrate the cytoplasm. The measured impedance of the cell suspension exhibits the electrical properties of the plasma membrane and cytoplasm at lower and higher frequencies, respectively. Therefore, the electrical impedance |Z| and capacitance Cp were measured at 10 kHz, 100 kHz, and 1 MHz in this study.To evaluate the electrical properties of the cells, an equivalent circuit  was intr6 cells/mL. Afterward, the cell suspension (300 μL) was injected into the PDMS reservoir, and the electrical impedance and capacitance at 100 kHz were measured at 0, 60, 120, 240, 480, and 720 s after the injection of the cell suspension. To determine the appropriate cell concentration for the impedance measurement device, the electrical impedance measurements of cell suspensions with different cell concentrations were performed without DEP cell accumulation. As the representative of differentiated and de-differentiated chondrocytes, chondrocytes passaged twice (P2) were prepared and suspended in a LC buffer at concentrations of 0.1, 1.0, and 10 × 10p–p at 1 MHz was applied to cause positive DEP on the chondrocytes [To evaluate cell accumulation by the positive DEP force, impedance measurements were performed after cell accumulation for 0, 60, 120, and 180 s. The cells were sedimented in the reservoir for 720 s before the impedance measurements and DEP experiments. An AC voltage of 10 Vdrocytes , and the6 cells/mL. The cell suspension (300 μL) was injected into the PDMS reservoir, and the cells were sedimented for 720 s before impedance measurements. The electrical impedance and capacitance were measured at 10 kHz, 100 kHz, and 1 MHz immediately after cell accumulation by positive DEP at a voltage of 10 Vp–p at 1 MHz for 180 s.The electrical impedance and capacitance of differentiated and de-differentiated chondrocytes were measured to verify the effectiveness of the proposed method in identifying chondrocyte phenotypes. According to the previous studies , de-diffn = 5–7) were analyzed, and each data point represents the mean and standard deviation. The statistical significance of the experimental data was evaluated using Bonferroni’s test.Most of the data are representative of three individual experiments from different calf donors with similar results. For each experimental group, five to seven samples (6 cells/mL chondrocyte suspensions were measured. The chondrocytes sedimented on the micro-electrodes according to the experimental time and impedance of 1.0 and 10 × 106 cells/mL chondrocyte suspensions decreased with increasing experimental time and reached a plateau after 480 s, whereas that of the 0.1 × 106 cells/mL suspension slightly changed during the experimental period and had a similar value to that of the LC buffer without chondrocytes (6 cells/mL without dielectrophoretic cell accumulation.To determine the appropriate cell concentration for our impedance measurement device, the electrical impedances of 0.1, 1.0, and 10 × 10drocytes . In thisElectrical impedance measurements were performed after cell accumulation by DEP force. During DEP, chondrocytes were accumulated on the microelectrodes and formed pearl-chain-like clusters, which bridged the adjacent electrodes . We prevTo verify the possibility of evaluating the de-differentiation phenotype of chondrocytes, electrical impedance measurements were performed on chondrocytes at different passage numbers. The chondrocytes were de-differentiated in an in vitro conventional monolayer culture . PrimaryThe electrical impedance of the chondrocytes decreased with increasing passage number at 10 kHz, whereas the capacitance increased . The impWozniak et al. reported that the elastic modulus of chondrocyte plasma membranes decreased with an increase in passage number . FurtherIn this study, we proposed electrical impedance measurements for living cells supported by DEP cell accumulation. To verify the proposed method, we developed an impedance measurement and DEP device using transparent opposing comb-shaped electrodes. Appropriate cell concentrations and DEP conditions were determined using bovine chondrocytes. Based on the results of this study, the measurement sensitivity of the device was improved by DEP cell accumulation. Furthermore, differentiated and de-differentiated chondrocytes were identified by measuring their electrical impedance and capacitance.


Cyclotella meneghiniana was the most dominant species in three seasons and three areas. The results of random forest are generally consistent with the results of CCA in spring, when the main environmental factors affecting phytoplankton were NTU and WT; the result in summer and autumn agreed with those of CCA, which awaits further study. In addition, the phytoplankton is mainly affected by WT, depth, and DO in the lake area, TP, DO, and NTU in the river area, and WT in the wetland area.Phytoplankton, as the primary producer of the wetland water ecosystem’s food chain, are very sensitive to environmental changes. In order to explore the significance of phytoplankton in protecting ecosystem integrity, the wetland ecosystem in Zhalong wetland, one of the most important international wetlands, was selected as the research area. For the study, 81 sampling sites were set up in the whole wetland, and phytoplankton samples and water quality environmental factors were measured in spring, summer, and autumn of 2019. The phytoplankton community structure and water environmental factors were evaluated by canonical correspondence analysis (CCA). The main research findings are as follows: a total of 292 species and variants of phytoplankton belonging to 8 phyla and 110 genera were identified within Zhalong wetland in spring, summer, and autumn 2019. The total phytoplankton abundance and biomass in summer were higher than in spring and autumn, and  Phytoplankton, as primary producers in the wetland hydrosphere ecosystem, have rapid reproduction and are sensitive to environmental changes. The species composition abundance and the dominant species are important community structure characteristics and critical indicators reflecting water quality ,2. They 2) lies between 46°52′–47°32′ N and 123°47′–124°37′ E  ,17. HoweAccording to the regional climate and ecological environment characteristics of Zhalong wetland, phytoplankton, and environmental factors were collected in May (spring), August (summer), and October (autumn) 2019, and 81 sampling sites were set up in lake, wetland, and river. The sampling sites in the three seasons were the same: lake area (L1~L53), where natural lakes are full of aquatic vegetation and aquaculture; wetland area (W1~W21), where the dominant vegetation group of Zhalong wetland is reed and cattail; river area (R1~R7), where the sites are mainly distributed in Wenghai drain, and there is substantial high overlap with human-disturbed environments.First, three duplicate quantitative samples of phytoplankton (1 L) were collected according to the following parameters: (a) at a water depth below 3 m, the sample was collected from 0.5 below the surface; (b) at a water depth of 3–10 m, mixed samples were taken at the surface and bottom layers; (c) at a water depth more than 10 m, mixed samples were taken at the surface and middle and bottom layers. Collection at each site was conducted using a phytoplankton net , with horizontal hauls in the subsurface (0.5 m depth). Each 1 L phytoplankton sample was preserved in Lugol (1%) and formaldehyde (5%) for later identification and enumeration. A light microscope (MOTIC BA410) was used for applied to obtain the species and quantity of phytoplankton (V = 0.1 mL), which were identified to the lowest taxonomic level using keys by described in ,20.−), pH, ammonium nitrogen (NH4+-N), conductivity (EC), turbidity (NTU), and dissolved oxygen (DO). Water transparency (SD) was measured using a Secchi disc. Water samples were taken at a depth of 0.5 m below the surface, stored in 1 L PE bottles, and analyzed for total nitrogen (TN), total phosphorus (TP), and chemical oxygen demand (CODcr); the method referred to water and wastewater monitoring method IV [Seven environmental factors were recorded using a multiparameter probe (YSI6600) in the field: water temperature (WT), chloride ion  of selected phytoplankton species were normalized using the formula lg(1 + x) ,23 to maCCA is an ordination analysis that connects environmental variables and community composition so that the variables can be used to interpret the composition pattern of the community structure at study sites ,25, and The random forest (RF) model is capable of handling nonlinear data well and has significant advantages in characterizing interactions between variables. Furthermore, it is insensitive to unbalanced and missing data and has import efficiency in processing with regression and classification. RF method was implemented in the randomForest package in R. The R software package, a port of Breiman and Cutler’s  originalDuring the investigation period, a total of 292 species within Zhalong wetland belonging to 110 genera were detected among the samples, including Cyanophyta (19 species), Bacillariophyta (103 species), Chrysophyta (nine species), Xanthophyta (one species), Pyrrophyta (six species), Cryptophyta (four species), Euglenophyta (31 species), and Chlorophyta (119 species).In the whole region, the dominant species were Chlorophyta, Bacillariophyta, and Euglenophyta, comprising 86.64% of the total. Among them, Chlorophyta accounted for the largest proportion with 40.75%, followed by Bacillariophyta with 35.27%, Euglenophyta with 10.62%, Cyanophyta with 6.51%, Chrysophyta with 3.08%, Pyrrophyta with 2.05%, Cryptophyta with 1.37%, and Xanthophyta with 0.34%.There are more phytoplankton species in spring (May) than in summer (August) and autumn (October). In autumn, low water temperature becomes the limiting factor for the growth of phytoplankton rather than nutrients. Affected by the flood disaster in 2019, many rivers in Heilongjiang Province exceeded an alert level in summer. As a result, the water level of rivers and lakes within Zhalong wetland rose significantly, and the turbidity of water bodies also increased greatly, resulting in fewer phytoplankton species. Overall, there were fewer species in Pyrrophyta and Chrysophyta, mainly Glenodinium and Dinobryon, for which weak acidic conditions with less organic matter content and excellent water quality are suitable for growth.In the entire Zhalong wetland, 138 species of phytoplankton belonging to seven phyla and 66 genera were found at sampling points in the river area, including Cyanophyta , Bacillariophyta , Chrysophyta , Pyrrophyta , Cryptophyta , Euglenophyta , and Chlorophyta . Of the 138 phytoplankton species, 86 species were present in spring, along with 71 in summer, and 62 in autumn.A total of 181 species of phytoplankton, belonging to eight phyla and 82 genera were found at sampling points in the wetland area, including Cyanophyta , Bacillariophyta , Chrysophyta , Pyrrophyta , Cryptophyta , Euglenophyta , Chlorophyta , and Xanthophyta . Of the 181 phytoplankton species, 97 species were present in spring, along with 122 in summer and 113 in autumn.A total of 245 species of phytoplankton, belonging to eight phyla and 103 genera were found at sampling points in the lake area, including Cyanophyta , Bacillariophyta , Chrysophyta , Pyrrophyta , Cryptophyta , Euglenophyta , Chlorophyta , and Xanthophyta . Of the 245 phytoplankton species, 181 species were present in spring, along with 176 in summer and 140 in autumn.4 to 6560.4 × 104 cells/L, and the average abundance at each sampling point in the river, wetland, and lake areas is shown in The abundance of phytoplankton at each sampling point ranged from 38.4 × 104 cells/L; the largest share was Chlorophyta (60.23%), followed by Bacillariophyta (27.05%), Cryptophyta (3.60%), Euglenophyta (3.54%), Cyanophyta (3.15%), Chrysophyta (1.96%), Pyrrophyta (0.46%), and Xanthophyta (0.01%).The total abundance of phytoplankton within Zhalong wetland was 66,036.4 × 10Cyclotella meneghiniana was the highest (2425.2 × 104 cells/L), followed by Synedra acus (2085.6 × 104 cells/L); in summer, the abundance of Chlorella vulgaris was the highest , followed by Cyclotella meneghiniana (1764 × 104 cells/L); in autumn, the abundance of Cyclotella meneghiniana was the highest (1956 × 104 cells/L), followed by Chlorella vulgaris (1516.8 × 104 cells/L). The latter two are widely distributed species and have always been the dominant species in the Zhalong wetland [Cyclotella meneghiniana can be high. It is more than helpful for it to become the competitive species in natural water bodies, as it can still reproduce slowly at low temperatures (about 10 °C). Chlorella vulgaris mostly grows in small fertile water bodies and is common in lakes, ponds, and shallow harbors.In spring, the abundance of  wetland . Under sThe phytoplankton biomass varied between 0.70 and 107.49 mg/L at the sampling sites; the average biomass of each sampling site in the river, wetland, and lake areas is shown in Synedra acus, Cyclotella meneghiniana, and Ceratoneis Closterium, two in Chlorophyta: Ankistrodesmus angustus and Chlorella vulgaris, and one in Cryptophyta: Rhodomonas lacustris.There were four dominant species in lake areas, with dominance between 0.024 and 0.418, seven dominant species in river areas, with dominance between 0.020 and 0.285, and nine dominant species in wetland areas, with dominance between 0.031 and 0.120. In comparison, Sanjiang Plain  ,31, is dp < 0.05) except water transparency (SD) (p > 0.05).A total of 12 environmental factors were evaluated in the survey . One-waycr, Cl− and NH4+-N changed significantly. Due to the purification effect of submerged vegetation and emergent vegetation on water quality, the average value of TN in wetland areas was the lowest, and, in lake areas with high nutrients, the average value of total nitrogen was high. The mean value of CODcr was the lowest in lake areas and the highest in river areas; WT was not significant in spatial variation. The average value of Cl− was the highest in lake areas. There are many lakes within the Zhalong wetland, most of them stocked with fish, which would lead to a high Cl− content.It can be observed from p-value > 0.05 (0.762); hence, they were not included in the environmental factors for CCA. The environmental factors selected for CCA were TN, TP, CODcr, WT, Cl−, pH, NH4+-N, EC, NTU, DO, and depth, and the chosen phytoplankton were species with an occurrence frequency of ≥10% during the sampling period, i.e., 54 phytoplankton species  TN and TP had remarkable effects on phytoplankton in autumn. NH4+-N is also an important environmental factor affecting the growth of phytoplankton in autumn.The ordination plot of the species–environment relationship revealed the following: (1) NTU, TN, and WT had a significant influence on the growth of phytoplankton in spring. The species are projected on the extension line of the environment vector; the species closer to the arrow of the environment vector were the most competitive when the environment changed significantly, and vice versa. Several phytoplankton species appear on the environment vector arrows in spring, indicating that fewer species were tolerant to a single environmental factor. (2) The phytoplankton showed a significant relationship with NTU in summer. The rainy season is in summer, and the influx of a large amount of sediment makes the water turbid and interferes with the growth of phytoplankton; therefore, NTU is inversely proportional to phytoplankton. NHThe CCA of phytoplankton in lake, river, and wetland areas showed that 11 environmental factors explained 29.1%, 63.6%, and 31.0%, respectively, of the total variance of species distribution. The biplots  indicatecr, WT, Cl−, pH, NH4+-N, EC, NTU, DO, and depth) explained 26.22% of the total variance. cr (%IncMSE = 4.594), TN (%IncMSE = 4.244), and DO (%IncMSE = 2.329) as the important variables influencing phytoplankton in spring; these five factors explained 29.86% of the total variance, which was higher than the previous value (26.22%). These results are generally consistent with those of the phytoplankton CCA in spring. The relationship between environmental factors and phytoplankton is not a simple linear relationship. A nonlinear model (random forests) can better handle such cases. The line chart of the correlation between five main environmental factors and phytoplankton based on random forest , NTU (%IncMSE = 15.79), depth (%IncMSE = 6.090), WT(%IncMSE= 3.725), NH4+ -N (%IncMSE = 3.245), TN (%IncMSE = 2.024), and DO (%IncMSE = 1.870) as the important variables influencing phytoplankton in summer, explaining 53.63% of the total variance, which was higher than the previous value (52.19%). There was a deviation in the random forest result from that of CCA in summer.The 11 environmental factors explained 52.19% of total variance in summer, and the input feature importance ranking is shown in − was less than 200 mg/L, it had a minor effect on species abundance, but when Cl− concentration was more than 200 mg/L, the phytoplankton abundance growth appeared stepwise on the line; the abundance increased rapidly when NTU > 60, TN > 3 mg/L, and WT > 26 °C, and there was a negative correlation between phytoplankton abundance and water depth.A line chart of the correlation between the seven main environmental factors and phytoplankton in summer  shows th4+ -N (%IncMSE = 2.064), Cl− (%IncMSE = 2.040), WT (%IncMSE = 1.112), depth (%IncMSE = 0.852), and EC (%IncMSE = −1.903), explaining 29.05% of total variance, which is higher than previous value (25.10%).The 11 environmental factors explained 25.10% of the total variance in autumn, and the input feature importance ranking is shown in As shown on the line chart for autumn , the phyThe random forest model for phytoplankton and environmental factors appeared to have a poor fit to the data in each study area. After cross-validation, the total percentage of variance explained by the random forest model with environmental factors decreased from 16.65% to 10.27% for lake areas, and from 38.7% to 21.37% for river areas, while a negative value (−14.86%) was obtained for wetland areas. It was observed that the random forest model was not applicable to correlation analysis between phytoplankton and environmental factors within the Zhalong wetland area. This may be due to the overfitting of the model to the details of the noise in the dataset. That is, the training and test sets showed tremendous variability in the dataset. However, the specific mechanism needs to be studied further.Cyclotella meneghiniana and Synedra acus (2425.2 × 104 cells/L and 2085.6 × 104 cells/L respectively), with larger individuals; consequently, the highest average biomass was observed in the river area. Thermophilic species increased rapidly in the lake area in summer, such as Chlorella vulgaris, which was absolutely dominant in abundance , but the individuals were so small that they did not dominate in biomass. Zhalong wetland has four distinct seasons and prominent climate characteristics; Zhalong Wetland has a diverse, variable ecosystem with a variety of phytoplankton species. With reference to the previous studies in the Zhalong wetlands, as well as the results of this thesis, abundance and biomass of phytoplankton varied significantly with seasons [4+-N was an essential restrictive environmental factor. According to the results of CCA for autumn, TN and TP were the main environmental factors limiting the growth of phytoplankton. CCA indicated that the main environmental factors affecting phytoplankton abundance were WT, TN, and NTU in lake, river, and wetland areas, respectively. Compared to river areas and wetland areas, several lakes in the Zhalong Wetland have much higher amounts of TP and TN, and these lakes are small, confined water bodies. Additionally, the water body is seriously eutrophicated due to a lack of emergent aquatic plants, a small surface area, and weak self-purification capabilities. As a result, the phytoplankton community structure in the lake region is greatly impacted by NH4+-N. In summary, WT, nitrogen, and phosphorus nutrients were the most critical environmental factors for phytoplankton in Zhalong wetland.In a temperate climate, Chlorophyta is dominant at high nutrient levels in early spring and summer, while Bacillariophyta is dominant at low temperatures. Therefore, within Zhalong wetland, the largest numbers of Chlorophyta were found in spring and summer 2019 (41.95% and 45.73%), and the largest numbers of Bacillariophyta were found in autumn (39.63%). Nutrients are generally considered to be the most important driving factors for the growth of phytoplankton. They mutually affect and transform each other ,34. With seasons . The CCABefore the implementation of ecological water replenishment in Zhalong wetland, except for annual precipitation, there was almost no external water supply; the Wuyuer and Shuangyang rivers were the primary water sources. The construction of agricultural small ponds and reservoirs upstream intercepted two-thirds of the water of Wuyuer River and almost all the water of Shuangyang River, thus blocking the flow into Zhalong wetland. After introducing the Nenjiang River as a water source for ecological water replenishment, the sluice is opened in June every year, which alleviates the problem of water shortage in Zhalong wetland to a certain extent. In the rainy season, rainwater and flood peaks caused by opening the gate lead to domestic sewage and farmland fertilizer being washed into the wetland, increasing the nitrogen and phosphorus nutrients in the region’s water, which is a main advantage for phytoplankton in Chlorophyta.− was the most important environmental factor affecting phytoplankton, not NTU, but it was the second most important environmental factor in CCA. There were also some deviations between the random forest method and CCA results for autumn, although total TN was equally important in both methods. In studying the correlation between phytoplankton abundance and environmental factors, the partialPlot diagram showed that (1) increased of TN and DO in spring would promote an increase in phytoplankton abundance; (2) in summer, NTU, TN, and WT had no significant effect on increasing phytoplankton abundance within a specific range; however, but when the values exceeded a specific range, abundance increased rapidly, showing a correlation with water depth; (3) in autumn, phytoplankton abundance increased with increasing NTU. Nevertheless, in evaluating the correlation between phytoplankton and environmental factors in each study area , compared with CCA, the random forest model revealed its lack of adaptability.In this paper, using the random forest model to study the critical water environmental factors affecting the growth of phytoplankton, the results for spring are consistent with those of CCA, whereas the results for summer showed a deviation between the two. In the random forest analysis, ClResearch on phytoplankton in Zhalong wetland is focused on the influence of different environmental gradients on phytoplankton biomass, diversity, and community structure distribution ,36. Two In this study, the phytoplankton population in the Zhalong wetland was dominated by Chlorophyta, with a total proportion of 40.75%, followed by Bacillariophyta, with 35.27%. The dominant phytoplankton species varied from Bacillariophyta to Cyanophyta and Chlorophyta depending on spatiotemporal variation in Zhalong wetland. We selected 54 species of phytoplankton (occurrence frequency ≥ 10%) combined with water environmental factors that showed significant differences in each season after one-way ANOVA to analyze the influence of water environmental factors on the phytoplankton community. CCA results showed that NTU, TN, WT, and TP had a remarkable effect on phytoplankton within Zhalong wetland. The results of random forest regarding the main environmental factors affecting phytoplankton in spring showed good agreement with the results of CCA. The top two environmental factors are turbidity (NTU) and water temperature (WT); however, the results of random forest deviated from those of CCA in summer and autumn. In conclusion, the most important environmental parameters for phytoplankton in Zhalong Wetland were WT, nitrogen, and phosphorus nutrients. Among them, TP and TN were probably the main control environmental factors to further eutrophication problems. Our findings suggest that managers of the Zhalong wetland should optimize the upstream water allocation, enhance the internal ecology of the wetland, and preserve its function as the “Earth’s kidney”.