Few studies have examined the optimal adjustment indices for predicting low muscle strength or physical performance in hemodialysis (HD) patients. Thus, the present study aimed to identify optimal adjustment indices for predicting strength and/or physical performance in HD patients.2 (Ht2), body surface area, or body mass index. Physical performance tests , and short physical performance battery) were also evaluated. Participants with a below median value for each physical performance test were defined as the low group.Our study was performed at an HD center (n = 84). Appendicular lean mass  was calculated using dual-energy X-ray absorptiometry. ALM were adjusted to body weight, height2 values between the low and normal physical performance group in all physical performance tests except short physical performance battery. The multivariate analysis revealed a significant difference in ALM/Ht2 between the low and normal physical performance groups in hand grip strength, 5 times sit-to-stand test, sit-to-stand for 30-second test, and AST. In women on HD, most indices were not associated with physical performance or strength.The mean participant age was 55.6 ± 12.8 years; 44 (52.4%) were men. The univariate analysis revealed a significant difference in ALM/Ht2 may be the most valuable among various variables adjusted for ALM for predicting physical performance or strength.We demonstrated that, in men on HD, ALM/Ht These lead to sarcopenia defined as decreased muscle mass and/or decreased physical performance.–8 This is associated with high morbidity and mortality rates among HD patients.–12 Therefore, the early prediction and intervention of decreases in muscle mass are essential to improving the prognosis of HD patients.Chronic kidney disease is a common public health problem, and chronic kidney disease can progress to end-stage renal disease requiring dialysis or transplantation. Hemodialysis (HD) is the most commonly used treatment modality in patients with end-stage renal disease. Data from the United States Renal Data System and Korean registries showed that 87.9% of end-stage renal disease patients in the United States and 77% in Korea are on HD.–8 Because raw DEXA data do not consider body size, various adjustment indices using variables such as body weight (BW), body surface area (BSA), or body mass index (BMI) have been used. Previous studies showed an association between various variables such as adjusted index and physical performance or metabolic disturbances in the general population.–17 These showed different prevalence values of sarcopenia or abilities to predict physical performance among various variable-adjusted indices. However, differences among nations, ethnicities, and diseases should be considered. Few studies have examined the optimal adjustment indices for predicting low muscle strength or physical performance in HD patients. Thus, the present study aimed to identify optimal adjustment indices for predicting strength and/or physical performance in HD patients.In HD patients, a definite conclusion regarding optimal muscle mass measurements is lacking, but dual-energy X-ray absorptiometry (DEXA) is the most popular and acceptable method for predicting muscle mass.22.1Our study was performed at an HD center in the Republic of Korea between January and December at 2015 , serum albumin level (g/dL), dialysis vintage, and the presence of diabetes mellitus (DM). Kt/Vurea was calculated using Daugirdas formula. If a patient had a self-reported history and a medical record of a DM diagnosis or medication use, the patient was defined as having DM.Laboratory or demographic data collected at enrollment included sex, age, hemoglobin level (g/dL), Kt/2 , BSA (m2), or BMI (kg/m2). BSA was calculated using the DuBois formula. BMI was calculated as BW divided by Ht2.Muscle mass measurements were performed using DEXA. Measurements were performed after the midweek HD session. DEXA was measured using a Prodigy Advance instrument and performed by a technologist while each participant was in a supine position and clothed in a light gown . Appendicular lean mass  was calculated using the sum of both upper and lower extremities. ALM were adjusted to BW (kg), height2) was calculated using the total FM determined from the DEXA measurements and height squared. In addition, computed tomography  images of each participant were obtained at the mid-thigh level, specifically at the midpoint of a line extending from the superior border of the patella to the greater trochanter . The thigh muscle area  and thigh intermuscular fat area  were measured from the images using ImageJ software . Thigh muscle quality (%) was calculated using 100 × IMFA/(IMFA + TMA). Sarcopenia was defined as both low muscle mass and low strength .The fat mass (FM) index  and recorded for maximum strength during the 3 trials on the dominant hand. The average steps per day (AST) were calculated using a pedometer.In our study, physical performance data included the 5-times sit-to-stand test (5STS), sit-to-stand for 30-second test (STS30), 6-minute walk test (6MWT), timed up and go test (TUG), gait speed (GS), and HGS. These tests were performed using standard protocols. We also defined a low physical performance group for each physical performance test. Participants with a below median value for each physical performance test were defined as the low group.The short physical performance battery (SPPB) test was calculated using previously defined methods from the GS, 5STS, and balance test (scored between 0 and 12).2.4t test. A multivariate analysis was performed using analysis of covariance. Correlations between 2 continuous variables were assessed using Pearson or partial correlation analyses. Linear regression analysis was performed to assess the independent predictors of each physical performance. The multivariate analysis was adjusted for age and DM. The level of statistical significance was set at P < .05.SPSS version 21.0 was used to analyze the data . Categorical variables are expressed as both counts and percentages. Continuous variables are expressed as mean ± standard deviation or standard error. For continuous variables, means were compared using Student 33.12, ALM/BSA, and ALM/BMI values were 19.9 ± 3.0 kg, 30.4 ± 4.0%, 7.12 ± 0.82 kg/m2, 11.4 ± 1.2 kg/m2, and 0.85 ± 0.13 kg/kg/m2 in men, and 14.6 ± 2.3 kg, 25.6 ± 3.6%, 5.99 ± 0.77 kg/m2, 9.3 ± 1.0 kg/m2, and 0.63 ± 0.11 kg/kg/m2 in women, respectively . The mean 5STS, STS30, 6MWT, TUG, AST, GS, HGS, and SPPB values were 9.2 ± 8, 18.4 ± 6.1, 473 ± 121, 7.2 ± 2.2, 4660 ± 3160, 0.97 ± 0.22, 29.9 ± 7.2, and 10.9 ± 1.7 in men and 8.5 ± 2.6, 17.2 ± 5.2, 443 ± 103, 7.5 ± 1.8, 5178 ± 3762, 0.86 ± 0.16, 21.8 ± 4.8, and 10.9 ± 1.5 in women . In the male and female participants, the BMIs were 23.7 ± 3.4 and 23.7 ± 3.9 kg/m2 (P = .970), the FM indices were 5.7 ± 2.7 and 7.8 ± 2.9 kg/m2 (P = .001), the TMAs were 109.6 ± 21.6 and 83.6 ± 16.4 cm2 (P < .001), the IMFAs were 5.1 ± 3.7 and 6.0 ± 5.8 cm2 (P = .355), and the thigh muscle qualities were 4.4 ± 3.0% and 6.5 ± 5.4% (P = .030), respectively. The number of patients with sarcopenia was 13 (15.5%), 10 (22.7%) of whom were men and 3 (7.5%) were women (P = .072).The mean participant age was 55.6 ± 12.8 years . The associations between muscle mass indices and physical performance were more weakened in multivariate analyses than in univariate analyses. Regarding differences in indices according to groups, some indicators were significant in univariate analysis but not in multivariate analysis .The median 5STS, STS30, 6MWT, TUG, AST, GS, HGS, and SPPB values were 7.9, 18.0, 494, 6.5, 4087, 1.01, 30.0, and 12.0 in men and 7.8, 18.0, 466, 6.9, 4292, 0.89, 22.0, and 11.0 in women, respectively. For men, differences in indices are shown Table 3.32 and ALM/BSA were statistically significant in all physical performances except 5STS for ALM/Ht2 and STS30 for ALM/BSA. Partial correlation coefficients for ALM/Ht2 were statistically significant in GS, 6MWT, TUG, and AST. Those for ALM/BSA were statistically significant in GS, 6MWT, and AST. In women, most indices showed no statistical significance in the correlation analyses . Linear regression analyses showed similar trends with results from the correlation analyses .In men, correlation coefficients are shown Table P = .184 for TMA and P < .001 for IMFA), and in women, these were 0.468 and 0.507, respectively (P = .002 for TMA and P = .001 for IMFA).In men, the correlation coefficients between the FM index and TMA or IMFA were 0.204 and 0.710, respectively  compared with the 22.7% in the male participants. Consequently, 3 factors—the combination with small muscle mass and high muscular fat, correlation between muscle mass and fat mass, and low prevalence of sarcopenia—may be associated with the lack of a relationship between muscle mass indices and strength or physical performances in women.In our study, there were no significant associations between muscle mass indices and strength or physical performances in the female participants compared with those in the male participants. Kim et al Relatively small muscle mass and high insulin resistance combined with inter- and intra-muscular fat accumulation may be associated with the lack of a relationship between 2 variables in women.HD patients have high insulin resistance compared with the general population, and women have less muscle mass than men. compared mortality rates among muscle mass indices in HD patients in the USA and showed that all known muscle mass indices at the time were not better in association with mortality compared with physical performance, GS, and strength. The mean BMI in their study was 27.4 kg/m2 in men and 29.2 kg/m2 in women . Another study using the same cohort showed that the height-adjusted muscle mass index underestimates the prevalence of low muscle mass among obese patients and was poorer in predicting GS compared with the BW, BSA, and BMI-adjusted muscle mass indices. However, a Japanese study with HD patients with a mean BMI of 21.9 kg/m2 showed that height-adjusted muscle mass is associated with all-cause mortality. The BMI values in our cohort were closer to the values reported in the Japanese study than to the values reported in the American studies. These results revealed that BW or BMI-adjusted muscle mass index can be useful in predicting outcomes in HD patients with high BMIs or who are obese. The height-adjusted muscle mass index may be more useful in predicting outcomes in HD patients with normal or low BMIs. To the best of our knowledge, although there are no definite recommendations to date regarding the optimal adjusted muscle mass index to predict outcomes in HD patients, its use may be considered with the BW or BMI-adjusted index in cohorts with high BMIs and the height-adjusted index in cohorts with low or normal BMIs.Kittiskulnam et al,42–46 However, HD patients in our cohort were relatively stable, and categories using these values revealed that most of the patients were in the normal group. These resulted in a biased distribution of dependent variables according to the category of independent variables, which was associated with a decrease in the goodness of the regression model. Therefore, we defined the cut-off using the median value, which would be helpful in decreasing the biased distribution of dependent variables. Large sample sized studies that include patients with various conditions would be needed to secure the goodness of the regression model despite a biased distribution. Analyses using broad ranges of muscle mass, physical performance, and strength would be more informative. Fourth, analyses without the adjustment of additional confounding factors were inherent limitations of our study. We did not perform multivariate analyses using sufficient confounding factors. Most variables, such as hemoglobin, serum albumin, serum calcium, inflammation, or dialysis adequacy, may be associated with muscle mass or physical activities via direct or indirect pathways. Our study had a small sample size, and adjustment using many variables may lead to statistical non-significance. However, patients in our study were relatively stable without a wide variation of these variables, which may be helpful in attenuating these limitations.Our study has some limitations. First, it was performed in a single center and included a small number of patients. Second, we enrolled all participants who agreed to provide informed consent and did not calculate a proper sample size for statistical significance. Third, the low groups as a categorical variable for SPPB, GS, HGS, 5STS, STS30, 6MWT, TUG, AST were defined using the median value of each variable in our study. There were cut-off values for some indicators, such as SPPB, GS, HGS, 5STS, STS30, or TUG, as previous studies described.Our study had some strengths despite these limitations. It was performed using comprehensive evaluations, including body composition measurements (such as DEXA and computed tomography), strength, and physical performance tests . Although the association between these muscle mass indices and each indicator was already evaluated in previous studies, there are few studies to date on the association between muscle mass indices and outcomes, including these comprehensive measurements. In addition, our cohort included HD patients as a specific population, and we identified the differences in associations in terms of sex. These differences in associations in terms of sex between muscle mass indices and strength or physical performances have not yet been fully elucidated in HD patients. It is difficult to form a definite conclusion due to the small sample size, single center, and cross-sectional natures of our study. However, our study may be helpful in recognizing the necessity of further studies regarding the association between muscle mass indices and strength or physical performance in HD patients. A future large prospective study is warranted to overcome these limitations.52 may be the most valuable among various variables adjusted for ALM for predicting physical performance or strength.Here we demonstrated that, in men on HD, ALM/HtSeok Hui Kang analyzed and interpreted the patient data; and writing the manuscript. Jun Young Do and Ji-Hyung Cho performed generation and collection of data. Seok Hui Kang and Jun Chul Kim performed drafting and revision of the manuscript. All authors read and approved the final manuscriptConceptualization: Seok Hui Kang.Data curation: Jun Chul Kim, Jun Young Do, Ji-Hyung Cho, Seok Hui Kang.Formal analysis: Jun Chul Kim, Seok Hui Kang.Funding acquisition: Jun Young Do.Investigation: Seok Hui Kang.Resources: Ji-Hyung Cho.Validation: Ji-Hyung Cho.Writing – original draft: Seok Hui Kang.Writing – review & editing: Jun Chul Kim.


Structures of new compounds were established based on elemental analysis, 1H NMR, 13C NMR, and IR spectroscopic techniques. The obtained compounds were tested for their antioxidative and cytotoxic activities and results pointed to selective antiradical activity of palladium(II) complexes towards •OH and -•OOH radicals and anti-ABTS  cation radical) activity comparable to that of ascorbate. Results indicated the effect of C1 and C2 on the enzymatic activity of the antioxidative defense system. In vitro cytotoxicity assay performed on different carcinoma cell lines , and one healthy fibroblast cell line (MRC-5) showed a cytotoxic effect of both C1 and C2, expressed as a decrease in carcinoma cells' viability, mostly by induction of apoptosis. In vivo toxicity tests performed on zebrafish embryos indicated different effects of C1 and C2, ranging from adverse developmental effect to no toxicity, depending on tested concentration. According to docking studies, both complexes (C1 and C2) showed better inhibitory activity in comparison to other palladium(II) complexes.Two newly synthesized 4-hydroxycoumarin bidentate ligands (L1 and L2) and their palladium(II) complexes (C1 and C2) were screened for their biological activities,  Cancer is characterized by uncontrolled cell growth and represents one of the major causes of mortality according to the World Health Organization . Some ciIn the search for phytochemicals with a broad spectrum of biological activity and low toxicity, coumarins have attracted intense interest from many research groups. Coumarins, oxygen-containing heterocycles with typical benzopyrone scaffold, are naturally occurring compounds isolated from different plants, fungi, and bacteria . This clin vitro cytotoxic activity  and the bidentate ligands L1 and L2 .Initially, heterocyclic compound 3-acetyl-4-hydroxycoumarin (AHC) was synthesized, as previously described , in the s of AHC –27. To o1 and L2 . Each of3-(1-((3-Hydroxyphenyl)amino)ethylidene)chroman-2,4-dione (L1). White powder. Yield: 0.64 g (89%). Anla. calc. For C17H13O4N (Mr = 295.28) %: C, 69.15; H, 4.44; N, 4.74. Found: C, 68.75; H, 4.24; N, 4.76. IR (KBr) ν cm−1: 3228 ; 3064 (=CH); 2992, 2934, and 2724 (CH); 1675 (C=O); 1607, 1599, 1564, and 1488 (C=C); 1147 (C-O). 1H NMR  δ ppm: 2.57 , 6.77 , 6.79 , 6.87 , 7.28 , 7.31 , 7.35 , 7.65 , 7.95 , 9.99 , 15.42 . 13C NMR  δ ppm: 20.6 (C–2′), 97.3 (C–3), 112.5 (C–2″), 115.5 (C–4″), 116.2 (C–5″), 116.5 (C–8), 119.9 (C–5), 124.0 (C–6″), 126.0 (C–6), 130.6 (C–10), 134.6 (C–7), 137.0 (C–1″), 153.4 (C–3″), 158.5 (C–9), 161.7 (C–2), 175.8 (C–4), 180.5 (C–1′).3-(1-((4-Hydroxyphenyl)amino)ethylidene)chroman-2,4-dione (L2). White powder. Yield: 0.62 g (86%). Anla. calc. For C17H13O4N (Mr = 295.28) %: C, 69.15; H, 4.44; N, 4.74. Found: C, 68.76; H, 4.37; N, 4.72. IR (KBr) ν cm−1: 3345 (OH); 3219 (NH); 3068 (=CH); 2934, 2826, and 2696 (CH); 1687 (C=O); 1611, 1564, 1513, and 1484 (C=C); 1198 (C–O). 1H NMR  δ ppm: 2.55 , 6.89 , 7.20 , 7.28 , 7.30 , 7.64 , 7.94 , 9.91 , 15.28 . 13C NMR  δ ppm: 20.5 (C–2′), 97.1 (C–3), 116.2 , 116.5 (C–8), 120.0 (C–5), 123.9 , 125.9 (C–6), 126.9 (C–10), 127.2 (C–1″), 134.5 (C–7), 153.4 (C–4″), 157.5 (C–9), 161.8 (C–2), 175.7 (C–4), 180.3 (C–1′).Bis(3-(1-((3-hydroxyphenyl)amino)ethylidene)chroman-2,4-dione-palladium(II))complex (C1). Yellow powder. Yield: 0.048 g (45%). Anla. calc. For C34H24O8N2Pd (Mr = 694.53) %: C, 58.79; H, 3.48; N, 4.03. Found: C, 58.34; H, 3.75; N, 3.80. IR (KBr) ν cm−1: 3415 (OH); 3048 (=CH); 2939 (CH); 1681 (C=O); 1602 (C=N); 1560, 1479, and 1428 (C=C); 1161 (C–O); 526 (Pd–O); 457 (Pd–N). 1H NMR  δ ppm: 2.59 , 6.78 , 6.80 , 6.82 , 7.10 , 7.23 , 7.3 , 7.59 , 7.99 , 9.98 , 9.81 . 13C NMR  δ ppm: 24.0 (C–2′), 105.1 (C–3), 111.9 (C–2″), 113.9 (C–4″), 115.5 (C–5″), 115.7 (C–8), 117.4 (C–5), 123.5 (C–6″), 126.4 (C–6), 130.2 (C–10), 133.9 (C–7), 147.7 (C–1″), 152.3 (C–3″), 158.4 (C–9), 161.5 (C–2), 171.9 (C–4), 180.5 (C–1′).Bis(3-(1-((4-hydroxyphenyl)amino)ethylidene)chroman-2,4-dione-palladium(II) complex) (C2). Yellow powder. Yield: 0.049 g (46%). Anla. calc. For C34H24O8N2Pd (Mr = 694.53) %: C, 58.79; H, 3.48; N, 4.03. Found: C, 59.00; H, 3.51; N, 4.03. IR (KBr) ν cm−1: 3345 (OH); 3028 (=CH); 2938 (CH); 1670 (C=O); 1602 (C=N); 1547, 1508, 1477, and 1487 (C=C); 1199 (C-O); 527 (Pd-O); 462 (Pd–N). 1H NMR  δ ppm: 2.58 , 6.71 , 6.87 , 7.07 , 7.25 , 7.63 , 7.99 , 9.65 , 9.90 . 13C NMR  δ ppm: 24.3 (C–2′), 105.1 (C–3), 115.8 , 116.5 (C–8), 117.5 (C–5), 123.3 , 126.3 (C–6), 126.9 (C–10), 127.1 (C–1″), 134.5 (C–7), 152.3 (C–4″), 157.4 (C–9), 161.2 (C–2), 169.1 (C–4), 180.3 (C–1′).13C and 1H NMR spectra of the investigated compounds, GIAO  approach was used , resulting in the formation of palladium(II) complexes, C1 and C2  complexes was carried out hoping to obtain new biologically active compounds. The synthesis of two novel ligands was performed in two steps as presented in 1 and C2 .Structural features of the examined compounds were determined by nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy and elemental analysis , as well as DFT methods.1H NMR revealed/confirmed the aromatic structure of the newly synthesized ligands by the existence of several signals within the characteristic range of the chemical shift values, between 6.77 and 7.95 ppm. Singlets at 2.55 (L1) and 2.57 (L2) ppm can be assigned to protons of the methyl groups at C2′ of both ligands. The presence of OH groups at m-hydroxyaniline and p-hydroxyaniline appeared as broad singlets, positioned at 9.99 ppm (L1) and 9.91 ppm (L2). As expected, the peak of the proton belonging to the amine functional group appeared as a singlet positioned at the highest frequencies, at 15.42 and 15.28 ppm, respectively. The 1H NMR spectra of C1 and C2 complexes were deprived of signals originated from the amino group, which is evidence that, before coordination to palladium(II) ion, the ligands were deprotonated. The chemical shifts of other protons in complexes are similar to the corresponding ones in the ligands' spectra.Interpretation of the obtained NMR spectra was facilitated by the use of literature data in which, in addition to the application of NMR spectroscopy, the structure of the compounds, which are structurally similar to the compounds analyzed in this paper, was determined by X-ray crystallography –49. Spec13C NMR spectra, the chemical shifts for C atoms of the coumarin moiety, for both ligands, were in the range of 97.06–175.80 ppm, while the corresponding signals of the aminophenol ring were moved downfield in the interval of 112.5–153.4 ppm. Most download shifts were detected for C1′ because of its bridging position between electronegative nitrogen and coumarin moiety. The signal from the most shielded carbon of the methyl group was detected at 20.5 ppm. Comparison of 13C NMR spectra of ligands and complexes revealed differences in the chemical shifts of a C4 carbon atom, which are shifted downfield relative to the corresponding ones in the ligands' spectra. This is expected given the fact that during the coordination of ligands and palladium(II) ion, a new bond is formed between Pd and O, which is followed by the transformation/reduction of the carbonyl group C=O into C–O. Moreover, the transformation of the C3–C4 single bond into a double bond contributed to the greater chemical shift of the C3 atoms in the complexes. Also, as evidence of the formation of a bond between palladium(II) and nitrogen is a chemical shift of C2′. This carbon atom was shifted downfield by 3.6 ppm (C1) and 3.8 ppm (C2).In −1, was assigned. The IR spectra also revealed bands assigned to vibrations of the NH group, in the 3228–3259 cm−1 range, and to stretching vibrations of the lactone carbonyl groups in the range of 1675–1687 cm−1. As opposed to ligands, IR spectra of the synthesized complexes showed no bands that could be assigned to NH vibrations. In the given spectra, the band positioned at 1602 cm−1 was assigned to C=N stretching vibration just like bands at 526 cm−1 and 457 cm−1 assigned to stretching vibrations of Pd-O and Pd-N bonds.Analysis of the IR spectra of two ligands confirmed the presence of OH group to which a broad band, in the region 3228–3345 cm1H NMR and 13C NMR chemical shifts, obtained at DFT/B3LYP-D3BJ level of theory, are presented in Tables R) and relatively small values of AAE . The reduction percentage of the complex C1 (5μM) was approximately 62% (from 83.51 ± 4.14 a.u to 31.51 ± 0.79 a.u). Unlike C1 and C2, their corresponding ligands L1 and L2 demonstrated no activity towards •OH just like AHC even when applied at a concentration as high as 50 μM.The antiradical activity of the selected compounds toward •OOH adduct were obtained after the addition of the AHC and C1 complex in the concentration range of 1-50 μM. However, the addition of 20 μM C2 complex induced reduction of the DEPMPO-•OOH adduct by approximately 78%  has shown low and statistically insignificant antiradical potential  cation radical (ABTS) quenching was noticed after the addition of 50 otential . The antμM to 50 μM. The viability of cells was tested by MTS assay 24 h upon treatments. Low concentrations, 0.1, 0.5, and 1 μM, of all tested compounds, exhibited no cytotoxic effect on any cell line (data not shown). However, concentrations above 5 μM  of complexes C1 and C2 showed a significant cytotoxic effect with the most prominent effect on the MIA PaCa-2 cell line , melanoma (A375), pancreatic carcinoma (MIA PaCa-2), and one healthy fibroblast cell line (MRC-5) were subjected to treatments with either DMSO, as a negative control, AHC as a starting compound, derivatives L1 and L2, or the corresponding palladium(II) complexes C1 and C2. Five different concentrations were used ranging from 0.1 ell line . The sec-5 cells . It is iμM to 50 μM. In this study, a concentration of 5 μM was used, which in previous experiments led to a significant reduction in viability. Cells were treated with 5 μM of C1 and C2 and after 24 h collected for Annexin V-FITC assay. As presented in Since coumarin complexes C1 and C2 exhibited the highest cytotoxic effect, particularly against MIA PaCa-2 cells, given compounds were further tested to determine the type of cell death they caused on this particular cell line. In previously used cytotoxicity assays, we used concentrations ranging from 0.1 in vitro, were also examined in vivo by monitoring the development of the Danio rerio (zebrafish) model system. The zebrafish model, which develops many cancer types of comparable signaling pathways and morphology as in humans, allowed high-quality in vivo toxicological testing of new coumarin derivatives. A range of different concentrations has been used , and the effect monitored up to 120 hpf (hours postfertilization). At 50 and 100 μM concentrations of C1 or C2, the majority of treated embryos were prevented from hatching . Finally, at 1 μM of either C1 or C2, no increase in adverse developmental effects was detected compared to nontreated embryos , which additionally included the effect on MnSOD activity  was used to test the dependence of the cytotoxic effects  complexes were obtained. Although the new ligands did not show better antioxidant or cytotoxic effects than the parent molecule, AHC, their palladium(II) complexes did.•OH and ABTS radicals and no activity towards •OOH, C2 complex showed more pronounced activity towards both •OH and •OOH radicals and no ability to reduce ABTS. The reduction ability of C1 towards ABTS was comparable to that of ascorbic acid. The presented result suggested that C1 and C2 display significant radical scavenging effects on biologically relevant •OH and •OOH radicals that may help prevent or ameliorate oxidative damage. It is possible to assume the influence of palladium ion on the antiradical activity as a contributing factor. The exact and relevant reaction mechanism of their reduction abilities is in progress and will be published in the future as a detailed SAR study.Presented results on antiradical activity of C1 and C2 are in agreement with the literature data, related to the activity naturally occurring and synthesized coumarin-based compounds, by which the activity of the same scaffold, among others, varies with the number of hydroxyl groups . EPR expIn vitro results on cytotoxic effects of new compounds clearly demonstrated the potential of C1 and C2 to significantly reduce cells' viability, especially against pancreatic carcinoma MIA PaCa-2 cell line, mostly by inducing apoptosis  and 60% (C2). Obtained complexes are proved very potent in terms of their antiproliferative effect that is even more important having in mind that the same derivatives have shown significantly lower cytotoxic potential on fibroblast deriving from normal tissue (MRC-5 cells). The difference in response between carcinoma and healthy cells could be a base for further investigation and improvement of these compounds. The results are also in agreement with the literature data related to some coumarin-containing compounds showing specific antitumor activity against pancreatic carcinoma [poptosis . These rarcinoma , 53.In vivo experiments performed on the zebrafish model have shown the embryotoxicity of the complexes ranging from low to complete absence of toxicity, at concentrations of 5 μM and less. Further testing will be necessary to determine the optimal effective concentration that will induce specific anticancer activity but not embryotoxicity.Increased ROS load is a hallmark of any type of cancer , 55, andAs in the cytotoxicity assay, the effects of complexes on antioxidant defense pathway enzymes (AOD) deviated significantly from the effects of the parent compound AHC, demonstrating that complexes have new, different, properties. AHC showed interesting effects indicating that in cells, it acts like a prooxidant, with SOD-inducing activity. Increased CAT activity in healthy cells was again induced only by AHC, possibly a consequence of the elevated peroxide production.Recently, data have been published indicating that the increased activation of AOD actually confers cisplatin resistance in lung cancer cell lines . On the We have shown that at C1 and C2 concentration cytotoxic for cancer cells, enzymes of AOD are mostly spared or somewhat downregulated, in both cell lines treated. Notable is the finding that C1 induces the reduction of major detoxifying enzyme GST in the pancreatic cancer cell line, potentially making those cells more vulnerable to further treatment . In contThe C1 and C2 have similar, albeit not identical impacts on MRC5 and MIA PaCa-2 SOD enzymes. Notable reduction of both Mn-SOD and Cu-Zn SOD in both cell lines suggests that antioxidative capacity for superoxide radical is dampened by C1 and up to some extent by C2, with C1 having a more prominent unwanted effect in healthy control cells. In contrast to C1, C2 is active towards superoxide radical (as shown by EPR measurements), and therefore, it is possible to assume that C2 mitigates some negative effects through its antiradical activity. The AOD effects of C1, a more potent apoptosis inducer, were strikingly different than C2, lowering glutathione–related enzymes in cancer cells. Since in healthy cells, GR activity decreased as well, and C1 exhibited a cytotoxic effect on healthy cells , that is probably the effect that in future studies should be explored and mitigated. It is important to emphasize that the interaction with AOD enzymes is not the primary apoptosis-inducing mechanism in cancer cells, as demonstrated by the PLSR model, which indicates that the mechanism of cancer cells killing by C1 and C2 needs to be explored further. The specifically large effect on cancer lines with known NRF2 activated signaling and consistent reduction of MnSOD activity indicates that C1 and C2 might interact with some component of the SIRT3-NRF2 pathway. Recently published data on 7-hydroxy-3-(4′-methoxyphenyl) coumarin report on its affinity for SIRT3 and the ability to activate it and subsequently increase SOD2 activity . Based oFollowing known facts that the excess ROS stimulate tyrosine kinases to activate NRF2 and that in many carcinomas, including pancreatic carcinoma, there is an overexpression of various tyrosine kinase receptors and their ligands, docking studies identified family of tyrosine kinase receptors as a possible important drug target for tested coumarin derivatives and their corresponding palladium(II) complexes. Tyrosine kinase inhibitors target specific tumor pathways associated with carcinogenesis. Although the inhibition of tyrosine kinases has been viewed as a promising approach for the treatment of pancreatic cancer preliminary results of trials with tyrosine kinase inhibitors have been disappointing regarding the treatment of this specific cancer . There hin vitro potent antiproliferative effect against pancreatic carcinoma cells, can interact with tyrosine kinase receptors. Docking analysis implies that C1 and C2 bind RTK more effectively in comparison to their corresponding ligands . C1 enters significant interactions with RTK establishing three hydrogen bonds via the amino acid residues ASP641, ASN659, and ARG646, one alkyl-alkyl, and four π-alkyl interactions (Table Gbind) values and consequently show better inhibitory activity in comparison to other palladium(II) complexes [According to docking studies, coumarin complexes C1 and C2, exhibiting  Figures , which i Figures . The resns Table . Both C1omplexes –77. Furt•OH, •OOH, and ABTS radicals. In vitro cytotoxicity, tested on three different carcinoma cell lines  and one healthy fibroblast cell line (MRC5), was cell-type selective and dependent upon the concentration of the tested complexes. The mode of cell death induced upon the action of complexes pointed to the early or late apoptotic and necrotic phases. In vivo cytotoxicity tested on zebrafish embryos, dependent upon the concentration of AHC and tested complexes, varied from preventing hatching to several developmental effects including skeletal and cardiovascular abnormalities. Investigated complexes showed different effects on AOD enzymes while the docking analysis proved the possible interaction of complexes with tyrosine kinase receptors and better inhibitory activity of C1 in comparison with other palladium(II) anticancer complexes.In this study, two new 4-hydroxycoumarin bidentate ligands and their palladium(II) complexes have been successively synthesized, structurally characterized, and screened for biological activity. Obtained compounds showed selective antioxidant activity towards In conclusion, preliminary studies imply the effect of C1 and C2 on pancreatic carcinoma cells as specific and more toxic compared to the effect on healthy cells. These types of compounds could be a useful starting point in future drug development.


N6-methyladenosine (m6A), N1-methylguanosine (m1G), 5-methylcytidine (m5C), and 5-methyluridine (m5U) are representative 2′-O-methylation and base-methylation modified epigenetic marks of RNA. Abnormal levels of these ribonucleosides were found to be related to various diseases including cancer. Serum is an important source of biofluid for the discovery of biomarkers, and novel tumor biomarkers can be explored by measuring these ribonucleoside modifications in human serum. Herein, we developed and applied a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method to determine the content of monomethylated ribonucleosides in human serum. The developed method enabled sensitive and accurate determination of these monomethylated ribonucleosides. By applying this robust method, we demonstrated the presence of Gm and Um in human serum for the first time, and we successfully quantified m6A, Gm, m1G, Cm, Um and m5U in serum samples collected from 61 patients with breast cancer and 69 healthy controls. We discovered that the levels of Gm, m1G, Cm, Um and m5U in serum were all significantly decreased in breast cancer patients whereas m6A was increased. We performed receiver operating characteristic (ROC) curve analysis, and obtained highest area under curve (AUC) value when combining these six monomethylated ribonucleosides together. These results suggest that m6A, Gm, m1G, Cm, Um and m5U might have great potential to be novel biomarkers for detection of breast cancer in the early stage. In addition, this study may stimulate future investigations about the regulatory roles of monomethylated ribonucleosides on the initiation and development of breast cancer.RNA methylation plays a significant regulatory role in various of physiological activities and it has gradually become a hotspot of epigenetics in the past decade. 2′-O-methyladenosine (Am), 2′-O-methylguanosine (Gm), 2′-O-methylcytidine (Cm), 2′-O-methyluridine (Um),  Post-transcriptional modifications of RNA has great research prospects and RNA epigenetics/epitranscriptomics has been proposed . More th6A, m1G, m5C, m5U and so on. When the hydrogen on the 2′-hydroxyl (-OH) of the ribose moiety is replaced by a methyl group (-CH3), it will form 2′-O-methylation ribonucleosides (Nm) including Am, Gm, Cm and Um. M6A is the most predominant modification in mRNA, and the aberrant level of m6A modification has great connection with the tumorigenesis and development  were bought from Sigma-Aldrich . 2′-O-methyladenosine (Am), N6-methyladenosine (m6A), 2′-O-methylguanosine (Gm), 1-methylguanosine (m1G), 2′-O-methylcytidine (Cm), 2′-O-methyluridine (Um), 5-methyluridine (m5U) and isotopically labeled standards [D3]m6A, [D3]Um and [13C5]m5U were obtained from Toronto Research Chemicals . [13C15N2]Gm, [13C15N2]m1G and [13C5]Cm were synthesized according to literature . Methanol of HPLC grade was purchased from J.T.Baker . Formic acid (HCOOH) was bought from Fluka . Ammonium formate, malic acid and 5-methylcytidine  achieved by Empower Pro 6.0 software was applied for analysis. A Waters Acquity BEH Amide column  was applied for chromatographic separation. 4000 QTRAP mass spectrometer  was applied for MS detection. The mass spectrometer was equipped with electrospray ionization (ESI) positive ion mode. Multiple-reaction monitoring (MRM) was chosen to acquire data. Data acquisition and processing were controlled by Analyst 1.6.3 software.The Ethics Committee of Medical Research of the Second Affiliated Hospital, Zhejiang University School of Medicine (SAHZU) approved our study. A total of 69 healthy volunteers  and 61 patients with breast cancer  were recruited from SAHZU. All breast cancer patients had a diagnosis report with a pathological stage of stage I or stage II at SAHZU between June 2020 and December 2020. The exclusion criteria were as follows: 1) Co-suffering other malignant tumors. 2) Having received any type of treatment for tumors. 3) Suffering from metabolic diseases, kidney diseases or liver diseases. 4) Taking any drugs for a long time. All participating volunteers agreed the informed consent in advance. Then, the serum samples were collected in the early morning and reserved at −80°C.At first, 10 μl of isotope-labeled internal standards (IS) mixed solution was added into 100 μl serum samples which were naturally thawed in ice. And then 330 μl pre-refrigerated acetonitrile/methanol of 2:1 (v/v) was added. After vortexed for 60 s, let it stand at −20°C for 2 h and centrifuged at 13,000 rpm, 4°C for 15 min orderly, 352 μl of the supernatant were taken out and then drain under vacuum. Then, 80 μl acetonitrile/water of 9:1 (v/v) was used to redissolve the dried samples. After vortexed for 10 s, ultrasonicated for 15 s and centrifuged at 13,000 rpm for 15 min at 4°C, 70 μl of the supernatant fraction were sucked into the sample bottle for subsequent HILIC-MS/MS detection.2O containing 10 mM ammonium formate, 0.2% formic acid and 0.06 mM malic acid, and (B) acetonitrile containing 2 mM ammonium formate, 0.2% formic acid and 0.06 mM malic acid. The eight analytes were perfectly separated at a flow rate of 0.4 ml/min by the optimized LC gradient program as follows: 0 min, 94% B; 4 min, 94% B; 6.1 min, 75% B; 6.5 min, 94% B; 8 min, 94% B. The BEH Amide column was set at 40°C and the samples temperature was maintained at 4°C. 5 μl of sample was injected each time and each sample was measured twice. To minimize the interference of the mass spectrometer, a switching valve was used and the eluents from the column were introduced into the ion source during 1.0–6.5 min.The mobile phase was (A) HThe ion spray voltage was kept at 5.5 kV and the ion source temperature (TEM) was maintained at 550°C. Ion source gas 1 (GS1), ion source gas 2 (GS2) and curtain gas (CUR) were all set at 45 psi. The ion transitions of these eight ribonucleosides and corresponding isotope labeled internal standard (IS) were shown in 6A, Gm, m1G, Cm, Um and m5U at different concentrations , which were mixed with IS solution , [13C15N2]Gm (10 nM), [13C15N2]m1G (20 nM), [13C5]Cm (30 nM), [D3]Um (20 nM) and [13C5]m5U (100 nM)), were made and analyzed. The calibration curves could describe as y = ax + b, where y represents the peak area ratio of the analyte to the corresponding IS and x denotes the concentration of the analyte. The limit of detection (LOD) and limit of quantification (LOQ) of each ribonucleoside were obtained by analyzing standard solutions with a signal-to-noise ratio of three and ten, respectively.The standard working solutions of m6A , Gm , m1G , Cm , Um  and m5U  were prepared in triplicate and were measured on the same day and three consecutive days, respectively. The accuracy was described as the ratio of measured value to the theoretical concentration.For the purpose of evaluating intra-day and inter-day precision, the quality control (QC) samples at three different levels of m6A , Gm , m1G , Cm , Um  and m5U . After 10 μl of IS solution (same as described above) was added, the serum samples were treated and analyzed as mentioned above. The recovery (R) of each analyte was calculated by /added concentration × 100%.For the purpose of evaluating the recovery of extraction, the serum samples were added with three different levels of mThe matrix effect was estimated by using a slope comparison method. By adding different concentrations  standard solution and IS to the serum extracts, the calibration curve was obtained. The ratio value of its slope to the slope of the standard solution calibration curve was matrix effect.p value less than 0.05 was considered meaningful. The area under the curve (AUC) was acquired by receiver operating characteristic (ROC) curve analysis, and the optimal cut-off value of the methylation modification in serum for the diagnosis of breast cancer was determined by the Youden index (Youden index = sensitivity + specificity −1). Receiver operating characteristic (ROC) analysis was performed to evaluate the ability of monomethylated modifications to distinguish cancer patients from healthy controls.Statistical analyses were achieved through SPSS 24.0 software . The concentration differences of serum monomethylated modifications between healthy volunteers and breast cancer patients were accessed by Two-tailed Student’s t-test, where The optimization of chromatographic conditions is mainly achieved by optimizing the type of chromatographic column and the composition of the mobile phase. In order to acquire symmetrical peak shape and great separation effect in a short time, it is very important to choose a column with high separation efficiency. The chemical structures of these monomethylated ribonucleosides were illustrated in + ions were observed in full scan ESI-MS. Then, the collision induced dissociation (CID) experiment was used to calculate the ion transitions of all analytes. In MS/MS, the ribose group can be easily eliminated due to the cleavage of the C-N bond. Taking Am and [13C5]Am as examples, abundant [M + H]+ ions at m/z 282.1 and 287.1 were observed for Am and [13C5]Am, respectively. The [M + H]+ ion of Am lost 146 and 151 Da was lost from [M + H]+ ion of [13C5]Am. Therefore, ion transition m/z 282.1→136.0 and m/z 287.1→136.0 was used for the quantification of Am and [13C5]Am, respectively. The ion transitions of other ribonucleosides and corresponding IS were shown in For the purpose of optimizing the MRM parameters, the mass spectrometer analyzed the standard solution injected by the peristaltic pump. Abundant [M + H]R2 < 0.999) in appropriate analytical ranges, and equations were showed in According to the aforementioned method, the prepared calibration curve showed excellent linearities . In the serum of breast cancer patients, the concentration of m6A, Gm, m1G, Cm, Um and m5U were in the range of 1.45–7.01, 7.84–21.39, 16.59–34.41, 24.61–57.14, 13.83–39.07 and 108.20–226.84 nM respectively, and the average concentrations were 4.48, 11.55, 25.15, 37.31, 20.27 and 159.40 nM, respectively (n = 61). As illustrated in 1G, Cm, Um and m5U in serum were intensely decreased in patients with breast cancer compared to healthy controls , but the concentration of m6A in breast cancer patients was much higher than that in healthy volunteers (p < 0.0001).The detailed concentrations of m6A, Gm, m1G, Cm, Um and m5U as potential breast cancer biomarkers, receiver operating characteristic (ROC) curve was plotted. Based on the Youden index, the optimal cut-off values of m6A, Gm, m1G, Cm, Um and m5U were 3.76, 12.99, 25.92, 37.31,21.62 and 169.58 nM, which indicates that if the detection values break through these thresholds, it increases the risk of breast cancer. Trapezoidal rule was used to calculate area under the curve (AUC). As demonstrated in 6A, Gm, m1G, Cm, Um, m5U and CA153, respectively. Most of the AUC values of ribonucleosides were higher than that of CA153, implying there might be better correlation between the levels of m6A, Gm, m1G, Cm and m5U in serum and the incidence of breast cancer, compared with CA153. Moreover, when all these serum monomethylated ribonucleosides were combined, the AUC could reach 0.93, which suggested the sensitivity and specificity for breast cancer diagnosis were dramatically increased. These results indicated that lower levels of Gm, m1G, Cm, Um and m5U and higher levels of m6A could be regarded as potential diagnostic indicators for the screening of breast cancer.To verify the significance of m6A, Gm, m1G, Cm, Um and m5U were identified and quantified. Of note, to the best of our knowledge, this is the first time that Gm and Um were detected in human serum. Besides, we elucidated the differences in the contents of these monomethylated ribonucleosides between these two groups and our data, to some extent, indicated that these six modified ribonucleosides might play as potential indicators in the early detection of breast cancer.In our study, a fast, robust, sensitive and trustable HILIC-MS/MS method was established for analysis of monomethylated ribonucleosides. A total of 130 serum samples from two groups containing breast cancer patients and healthy control were analyzed. Six modified ribonucleosides including m


In this in vitro AD model, we found APP accumulated in MAM and mitochondria, and altered levels of proteins implicated in ER-mitochondria tethering, Ca2+ signaling, mitochondrial dynamics, biogenesis and protein import, as well as in the stress response. Moreover, we observed a decreased number of close ER-mitochondria contacts, activation of the ER unfolded protein response, reduced Ca2+ transfer from ER to mitochondria, and impaired mitochondrial function. Together, these results demonstrate that several subcellular alterations occur in AD-like neuronal cells, which supports that the defective ER-mitochondria crosstalk is an important player in AD physiopathology.Alzheimer’s disease (AD) is characterized by the accumulation of extracellular plaques composed by amyloid-β (Aβ) and intracellular neurofibrillary tangles of hyperphosphorylated tau. AD-related neurodegenerative mechanisms involve early changes of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) and impairment of cellular events modulated by these subcellular domains. In this study, we characterized the structural and functional alterations at MAM, mitochondria, and ER/microsomes in a mouse neuroblastoma cell line (N2A) overexpressing the human amyloid precursor protein (APP) with the familial Swedish mutation (APPswe). Proteins levels were determined by Western blot, ER-mitochondria contacts were quantified by transmission electron microscopy, and Ca Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder affecting more than 47.5 million people worldwide . It is c2+ homeostasis, increased endoplasmic reticulum (ER) stress, and mitochondrial dysfunction, which occur years before the appearance of the pathological hallmarks of AD [2+ signaling, apoptosis, redox status, proteostasis including autophagy, and the ER stress-induced unfolded protein response (UPR), as well as mitochondrial dynamics and bioenergetics [The disruption of MAM has been implicated in AD physiopathology , since iks of AD ,7,8. MAMks of AD ,10,11. Tergetics ,14,15,16ergetics ,18,19. 2+ transfer from the ER, energetic metabolism and ATP production, dynamics, and biogenesis concomitantly with the analysis of ER stress-induced UPR. The main goal of our study was to investigate the structural alterations at MAM using an in vitro model of AD, namely the mouse neuroblastoma cell line (N2A) overexpressing the APP familial Swedish mutation (APPswe). The impact of MAM alterations on several cellular stress responses as well as in mitochondria functioning was also evaluated, including Ca2+ signaling and stress response mediators were determined by Western blot (WB). ER-mitochondria contacts were quantified by transmission electron microscopy, and Ca2+ homeostasis and mitochondria function were analyzed using fluorescent probes and Seahorse assays, respectively.In order, to study the structural alterations at MAM and the impact of these on cellular stress responses and mitochondria function in AD, we used an in vitro cell model. We isolated MAM, microsomes, and mitochondria fractions from N2A-WT and N2A-APPswe cell lines and protein levels of ER-mitochondria tethers, Cav/v) fetal bovine serum (FBS) , 3.7 g/L sodium bicarbonate , 1% (v/v) non-essential amino acids , and 1 mM sodium pyruvate , as previously described [v/v) penicillin/streptomycin  or 0.4 mg/mL geneticin  for WT or APPswe cells, respectively. Both cell lines were cultured at 37 °C in a humidified 5% CO2–95% air atmosphere. The wild-type mouse neuroblastoma cell line N2A (WT) and the N2A-APPswe cell line, which stably overexpress human Swedish mutant APP KM670/671NL (APPswe) , were maescribed ,22. Cellg for 5 min at 4 °C. Homogenization of pellets was then performed gently with a glass/teflon homogenizer in isolation buffer, pH 7.4, composed of 250 mM sucrose and 10 mM HEPES for microsomes and MAM isolation, or 225 mM mannitol, 75 mM sucrose, 30 mM Tris-HCl, and 0.1 mM EGTA for crude mitochondria isolation, supplemented with a 1% cocktail of proteases inhibitors . The suspension was centrifuged eight times at 600× g for 5 min at 4 °C to remove cell debris and nuclei and obtain the total fraction (TF). For microsomes and MAM isolation, the TF was centrifuged at 10,300× g for 10 min at 4 °C, resulting in a supernatant (cytosolic and ER/microsomes fraction) and pellet (MAM fraction). The supernatant was centrifuged at 100,000× g for 60 min at 4 °C in a Beckman ultracentrifuge  to pellet the ER/microsomal fraction that was then resuspended in PBS. For MAM isolation, the pellet resulting from TF centrifugation was resuspended in 1 mL SHM solution (250 mM sucrose and 10 mM HEPES) and centrifuged at 10,300× g for 10 min at 4 °C. The resulting pellet was resuspended in 600 µL mannitol buffer A , loaded on top of a 30% (v/v) Percoll gradient, and centrifuged for 65 min at 95,000× g in a Beckman ultracentrifuge . The upper band (containing the MAM fraction) was collected, diluted in PBS containing 1 mM PMSF, and centrifuged at 6300× g for 10 min at 4 °C. The supernatant was centrifuged at 100,000× g for 30 min at 4 °C in a Beckman centrifuge , and the pellet (MAM purified fraction) was resuspended in PBS containing 1 mM PMSF. To obtain the crude mitochondria fraction, the TF was centrifuged at 7000× g for 10 min at 4 °C in a Beckman centrifuge , the pellet was resuspended in starting buffer , and centrifuged at 7000× g for 10 min at 4 °C. This procedure was repeated five times. The mitochondrial pellet was resuspended in starting buffer, centrifuged at 10,000× g for 10 min at 4 °C in a Beckman centrifuge , and the resulting pellet  was resuspended in MRB buffer . Subcellular fractions purity was analyzed by WB  BSA Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBS-T). Membranes were incubated with the primary antibody overnight at 4 °C and afterward they were washed with TBS-T and incubated with the secondary antibody at room temperature for 1 h. Membranes were developed using ECF substrate  in ChemiDoc Imaging System , and quantifications were performed with the Bio-Rad Image Lab Software 6.1. Primary and secondary antibodies utilized for WB are summarized in Protein concentration was determined with the Pierce BCA Protein Assay Kit . Loading buffer was added to samples that were then boiled and denatured. Sample proteins (30–50 µg) were separated using 7.5–15% SDS-PAGE, transferred to a PVDF membrane , and blocked in 5% (g for 5 min to form a pellet. Cells were fixed with 2.5% (w/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h. Afterwards, cells were washed in the same buffer and the post-fixation was performed using 1% (w/v) osmium tetroxide for 1 h. Then, the cell pellets were rinsed two times with buffer and distilled water and, for contrast enhancement, cells were incubated in 1% (w/v) aqueous uranyl acetate during 1 h. After washing with water, cell pellets were embedded in 2% (w/v) molten agar and dehydrated in ethanol (30–100%). Then, cell pellets were impregnated and included in Epoxy resin . After polymerization, ultrathin sections were obtained, and observations were carried out on a FEI-Tecnai G2 Spirit Bio Twin at 100 kV. We analyzed approximately 10 different cells per cell line (n = 3) and MAM was considered when the distance between ER and mitochondria, which was measured using the ImageJ program, was ≤25 nm. At this distance, Ca2+ transfer between both organelles occurs through the IP3R-GRP75-VDAC axis [WT and APPswe cells were collected and centrifuged at 1008× 2–95% air atmosphere for 24 h. Culture medium was discarded, cells washed in Krebs solution , and loaded with 100 nM MitoTracker Green probe  in Krebs solution for 30 min at 37 °C in a humidified 5% CO2. Then, cells were washed in Krebs solution and incubated with 15 µg/mL Hoechst 33342 (Molecular Probes) in Krebs solution for 5 min at same conditions. Cells were washed and live images of the cells (608 WT cells and 498 APPswe cells) were captured with the Zeiss LSM 710 Confocal Microscope  with 63× oil objective and the fluorescence intensity analyzed using ImageJ software. The value of corrected total cell fluorescence (CTCF) was calculated using the following formula: CTCF = integrated density of selected cell − (area of selected cell × mean fluorescence of background readings).For imaging experiments, cells were cultured on 18 mm glass bottom culture 12-well plate  coated with poly-lysine D and incubated at 37 °C in a humidified 5% CO2+ levels were measured with Fura-2/AM  and mitochondrial Ca2+ content was measured with Rhod-2/AM . Cells were cultured in 48-well plates  in triplicate and incubated at 37 °C in a humidified 5% CO2–95% air atmosphere. The next day, cells were washed with solution A  and incubated for 30 min with 2 µM Fura-2/AM in solution A supplemented with 0.1% (w/v) BSA or for 45 min with 10 µM Rhod-2/AM in solution A at 37 °C. Cells were rinsed with solution B  and Ca2+ levels were measured upon stimulation with 5 µM thapsigargin  or 100 µM histamine . The fluorescence signal was measured at 37 °C during 10 min (10 in 10 sec) at λEx = 340/380 nm and λEm = 510 nm for Fura-2/AM and λEx = 552 nm and λEm = 581 nm for Rhod-2/AM using a plate reader . Intracellular cytosolic Ca2–95% air atmosphere for 24 h. Culture medium was discarded and replaced by fresh medium containing 4 µM TMRE  and cells were incubated at 37 °C for 30 min. Subsequently, the supernatant was removed, cells were washed using PBS and the fluorescence signal  was measured at 37 °C using a plate reader . Mitochondrial membrane potential (Δψm) was evaluated using the fluorescent probe TMRE (tetramethylrhodamine ethyl ester perchlorate). Cells were seeded in 96-well plates  in triplicate and incubated at 37 °C in a humidified 5% CO2+ assays, respectively. The multi-well plates were placed on a plate shaker at low speed for 10 min and then on ice during 20 min. Finally, 100 and 200 µL BCA assay mix  were added for the TMRE and Ca2+ assays, respectively. After a 30 min incubation period, absorbance was measured at 562 nm with a Spectrofotometer Spectramax plus 384 . The protein content of each well was determined by aspirating the assay buffer, followed by addition of 10 and 20 µL RIPA buffer  for TMRE and Ca2–95% air atmosphere. The next day, cells were washed in unbuffered DMEM  supplemented with 25 mM glucose, 1 mM sodium pyruvate, 0.58 g/L L-glutamine, and 1% (v/v) non-essential amino acids for oxygen consumption rate (OCR) assay or in unbuffered glucose-free DMEM for extracellular acidification rate (ECAR) assay. Cells were incubated for 1 h at 37 °C in pre-warmed medium with pH adjusted to 7.4 and plates were loaded into a Seahorse XF24 . OCR was determined under the following conditions: Basal measurements; after 1 µM oligomycin  addition; upon injection of 1 µM FCCP  and, finally, after a mixture of 2 μM rotenone  plus 2 μM antimycin A  was injected. Rotenone  and antimycin A  inhibit mitochondrial respiration allowing to determine the non-mitochondrial OCR. To calculate the basal mitochondrial OCR, the non-mitochondrial OCR is subtracted from the OCR obtained before the addition of oligomycin . To evaluate the maximal respiratory capacity, we subtracted the non-mitochondrial OCR from the OCR following carbonyl cyanide-4-(trifluoromethoxy) phenyl hydrazone (FCCP) addition (disrupts ATP synthesis by dissipating the proton gradient). To obtain the spare respiratory capacity, we subtracted the basal mitochondrial OCR from the maximal respiratory capacity. The mitochondrial ATP production was assessed after oligomycin addition, by subtracting the non-mitochondrial OCR from the OCR value after oligomycin.Cells were seeded  in cell culture microplates (5 wells per cell line) provided by the manufacturer and incubated at 37 °C in a humidified 5% COIn the ECAR assay, after baseline measurements, the following injections were performed to each well under similar conditions: First, 25 mM glucose, then 1 µM oligomycin, and, finally, an injection of 100 mM 2-deoxyglucose (2DG) provided a non-glycolysis extracellular acidification rate. Cells were first placed in a glucose-free medium and the first injection consisted in 25 mM glucose. The glycolysis rate was obtained by subtracting non-glycolysis ECAR from the ECAR post-glucose. Then, oligomycin was added to the cells to suppress mitochondrial ATP production. The glycolysis capacity was determined by the difference between the post-oligomycin ECAR and post-2-deoxyglucose baseline ECAR. After the assays, buffer was aspirated, 30 µL RIPA buffer was added per well, and protein content was measured using BCA assay mix to determine the protein content per well. In comparison with the basal rates, the % of change was calculated dividing the measured value by the average value of baseline readings and normalized to the total protein content of each well. The OCRs are normalized to the WT cells mean.t-test and two-way ANOVA with Sidak post hoc correction for grouped analysis. In case of non-normality distribution, data were analyzed by the Mann–Whitney U-test to compare two groups and Kruskal–Wallis Dunn’s correction for multiple comparisons. Statistical significance was considered at p < 0.05.Statistical analysis was performed using the GraphPad Prisma 8.0.2 software  and presented as mean ± standard error of the mean (SEM). All experiments were independent assays. Data were tested for Gaussian distribution using the Shapiro-Wilk test. Differences between the two groups were analyzed using unpaired 2+ channel, associated with the inositol 1,4,5-trisphosphate receptor (IP3R) present at ER, interacts with the mitochondrial voltage-dependent anion channel 1 (VDAC1) through the glucose-regulated protein 75 (GRP75) and mitofusin 2 (MFN2), present in both ER and mitochondria homo- and hetero-oligomerizes with the mitochondrial mitofusin 1 (MFN1), to tether both organelles [p < 0.0794) in APPswe cells when compared with WT cells  (p = 0.0656) (p < 0.001) (p < 0.0001) in mitochondria fluorescence intensity was observed in APPswe cells when compared with WT cells  a,c. Sinc 0.0656) d,e and a< 0.001) f in APPsWT cells g,h. Addi2+ signaling, which in turn regulates mitochondrial function. Dysregulation of mitochondrial Ca2+ and metabolism have been associated with AD development [2+ levels were measured with the Fura-2/AM fluorescent probe following treatment with thapsigargin (TG), which blocks the ability of the cells to pump Ca2+ into the ER by inhibiting the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) and depletes ER Ca2+. We observed a significant increase in Ca2+ flux from ER to cytosol (p < 0.01) in APPswe cells  in Ca2+ flux from ER to mitochondria (p = 0.05) in APPswe cells following treatment with histamine that triggers Ca2+ release through IP3R (2+ uniporter (MCU) proteins by WB. VDAC1 is a protein present in the OMM involved in ER-mitochondria tethering by forming a ternary bridging complex with the OMM chaperone GRP75 and IP3R in the ER, as well as in Ca2+ signaling by regulating Ca2+ transfer from ER to mitochondria via IP3R [2+ must cross the MCU located in the inner mitochondrial membrane (IMM) [p < 0.05) and MAM (p < 0.05) fractions was observed in APPswe cells when compared with WT cells (p < 0.01) (p < 0.05) (p < 0.05) and microsomes (p < 0.01) isolated from APPswe cells  . A signiWT cells e,g. Furt < 0.01) f,i, whic < 0.05) f,j, and we cells e,h. Sig-oduction ,35. Desp2+ homeostasis, ATP production via oxidative phosphorylation (OXPHOS), and apoptosis. Thus, these organelles regulate cellular fate and mitochondrial dysfunction is one of the most prominent features in AD that occurs in the early stage of this disease [2+ flux from the ER, which we showed to be decreased in APPswe cells (p < 0.01) (p < 0.001) (p < 0.001) (p < 0.05) in APPswe cells  b. Additi< 0.001) c, showin< 0.001) d. In thewe cells e.p < 0.01)  f. Altoge < 0.01) h.p < 0.01) (p < 0.01)  was added to determine the non-glycolysis acidification rate g. We det < 0.01) h and a 3 < 0.01) i when cop < 0.05) (p-DRP1/DRP1 and FIS-1 (Mitochondria are dynamic organelles undergoing cycles of fusion (union of two mitochondria) and fission (division of one mitochondrion into two daughter mitochondria) to maintain their shape, distribution, and size. A balanced mitochondrial dynamics is required to ensure a proper mitochondrial function and response to cellular stress . Impaire < 0.05) a,d in thnd FIS-1 a–c,e in Mitochondrial biogenesis is a process by which mitochondria increase their number/mass  contribu2+ signaling, and upon stress activates the UPR to restore proteostasis. The UPR activation can be triggered by the accumulation of pathogenic misfolded proteins, disruption of intracellular Ca2+ homeostasis, defects in autophagy, oxidative stress, proteasome inhibition, and metabolic or mitochondrial dysfunctions [2+ homeostasis and MAM signaling and its downregulation inhibits mitochondrial Ca2+ uptake [p < 0.0001) and MAM fractions (p < 0.05) (p < 0.05) in APPswe cells  obtained from APPswe cells when compared with WT cells  when compared with WT cells  a,b. Due  < 0.05) c,d and awe cells c,e. MoreWT cells f, g, whiWT cells f,h. PCNAp < 0.0001) and MAM (p < 0.001) fractions (p < 0.01)  is essential for mitochondrial functioning, since it is required for protein transport into mitochondria, including for the internalization of APP and Aβ . Accumulractions f,i and o < 0.01) j,k in AP2+ regulation, increased ER stress, alteration in glucose metabolism, and mitochondrial dysfunction have been reported. These alterations may, in part, be attributed to the disruption of the MAM-related functions [It has been described that MAM-related functions are perturbed in several neurodegenerative disorders, such as AD, Parkinson’s disease and amyotrophic lateral sclerosis . In AD, unctions ,51,52. 2+ transfer; (3) alterations in mitochondrial dynamics and function. In the present study we characterized the structural and functional alterations in MAM and mitochondria and their effect on stress response mechanisms, in an in vitro model of AD, to support the role of MAM in AD and their potential as therapeutic targets in this disease. Overall, our findings demonstrate: (1) Decreased ER-mitochondria contacts and alteration of MAM’s composition; (2) changes in ER-mitochondria Ca2+ released by the ER [2+ flux, since we observed a decrease in mitochondrial Ca2+ levels after histamine stimulation , as well as different in vitro experimental conditions . Other authors demonstrate that mitochondrial Ca2+ overload and, consequently, cell death, is induced by Aβ oligomers [2+ levels via MCU in the APP/PS1 transgenic (Tg) mouse model. The mitochondrial Ca2+ overload can induce to the opening of the mitochondrial permeability transition pore (mPTP) and caspases activation, consequently leading to cell death. The same authors reported that blocking MCU with Ru360 prevents the increase of mitochondrial Ca2+ levels [2+ uptake, even in the absence of mitochondrial membrane depolarization [2+ influx. Moreover, human post-mortem AD brains show a downregulation of genes related to mitochondrial influx Ca2+ transporter, MCU, and its regulatory subunits, and an upregulation of genes involved in mitochondrial Ca2+ efflux pathways, SLC8B1 (encoding NCLX), suggesting an adaptive mechanism to prevent mitochondrial Ca2+ overload [2+ uptake in Mfn2−/− MEFs in response to IP3-mediated cytosolic Ca2+ transients was also reported [−/− MEFs as compared with control cells [2+ transfer between both organelles [2+ transfer depends not only on the distance between both organelles, but also on the expression level of mitochondrial Ca2+ uptake machinery [2+ signaling, and Aβ is capable to upregulate VDAC1 in human neuroblastoma cell line [2+-sensitive chaperone complex with BiP/GRP78, and associates with IP3R. Upon IP3R activation, which decreases Ca2+ concentration at MAM, Sig-1R dissociates from BiP/GRP78 in ER periphery, promoting Ca2+ signaling into mitochondria via IP3R [2+ homeostasis [2+ to mitochondria affects their metabolism contributing to mitochondria dysfunction [First, we analyzed the structural and functional alterations at MAM in APPswe and WT cells. APPswe cells recapitulate amyloid pathology through increased Aβ generation . ER-mitoy the ER ,58. The y the ER . We alsoy the ER . Possiblmulation d, despitn the ER b. Concerligomers ,61. Calv+ levels , which irization , suggestoverload . A reducreported ,58. Furtol cells . Howeverganelles ,64. In Machinery  and the achinery . We founell line . Its oveell line . Sig-1R via IP3R ,67. Sig-via IP3R . Sig-1R via IP3R . Despiteeostasis . Simultaeostasis ,72. A deeostasis . Howevereostasis . An increostasis  and hippeostasis . Under teostasis ,78. Thisfunction . Accordifunction .2+ stress stimulus [2+ uptake were observed [2+ dysregulation [Previously, a reduction in mitochondrial membrane potential in AD fibroblasts after thapsigargin-induced Castimulus  and the observed . We alsoobserved . It is dobserved . Accordiobserved . As prevobserved . Our resgulation . Furthergulation .2+ uptake and ATP production. However, alterations in MAM functional features, such as Ca2+ signaling, induce ER stress and UPR activation, so ER-mitochondria communication could also regulate stress responses and the UPR at different levels [3Rs at MAM controlling mitochondrial Ca2+ uptake [2+ signaling modulation because ERO1α interacts with IP3R potentiating Ca2+ efflux from ER to mitochondria at MAM [2+ release [2+ efflux from ER to mitochondria.The ER-mitochondria contacts are also involved in the ER stress-mediated cell death and UPR, because several ER co-factors and chaperones are present at MAM. Moreover, impairment in the UPR, apoptosis, and misfolded proteins accumulation are common features of AD . In the t levels . Besidest levels . PERK ist levels  with itst levels . Besides+ uptake . Under r+ uptake . We also+ uptake . This pra at MAM ,91,92. O release ,44. Ther3K)/protein kinase B (AKT) signaling pathways demonstrating its involvement in cellular signaling [We also observed an increase of PCNA levels in APPswe f,h. Thisignaling . In matuignaling . Furtherignaling  and its ignaling  and in Aignaling . Thus, PInternalization of APP and Aβ peptide by mitochondria was observed in vitro ,97, in hIn conclusion, our results suggest that the overexpression of the familial APP Swedish mutation causes the accumulation of APP in mitochondria and MAM, which can contribute to the structural and functional changes in these subcellular fractions, affecting the response to stress and inducing energy deficits. Together, our findings support the idea that the defective crosstalk between ER and mitochondria, mediated by MAM, plays a key role in AD physiopathology that may represent a relevant therapeutic target in this disorder.


Data analysis has indicated that training during HIGH elicited a greater increase in VO2max  and VMA  compared with CTRL and SAND. While increases in VO2max and VMA following SAND were greater  than those observed after CTRL. Moreover, EPO increased to a greater extent following HIGH (25.3 ± 2.7%) compared with SAND  and CTRL  with a greater increase (p < 0.01) following SAND compared with CTRL. Furthermore, HIGH and SAND elicited a greater increase  in Hb compared with CTRL. There was no difference in Hb changes observed between HIGH and SAND (p = 1.0). Finally, athletes lost 2.1 ± 0.4% (p = 0.001) more weight following HIGH vs. CTRL, while there were no differences in weight changes between HIGH vs. SAND (p = 0.742) and SAND vs. CTRL (p = 0.719). High-altitude training and sea-level training on sand resulted in significant improvements in EPO, Hb, VMA, and VO2max that exceeded changes in such parameters following traditional sea-level training. While high-altitude training elicited greater relative increases in EPO, VMA, and VO2max, sand training resulted in comparable increases in Hb and may prevent hypoxia-induced weight loss.Altitude training increases haemoglobin, erythropoietin values among athletes, but may have negative physiological consequences. An alternative, although less explored, that has the potential to positively influence performance while avoiding some of the negative physiological consequences of hypoxia is sand training. Ten endurance-trained athletes  performed three 21-day training camps at different locations: at a high altitude (HIGH), at the sea-level (CTRL), at the sea-level on the sand (SAND). Differences in erythropoietin (EPO) and haemoglobin (Hb) concentration, body weight, VO High-altitude training has long been considered to improve the performance of endurance athletes . As the 2max over the 6-weeks was reported in the sand running group but not in the road running group. Therefore, sand has the potential of providing not only a unique training stimulus for athletes but also a viable option for recovery sessions.An alternative, although less explored, practice that has the potential to positively influence performance while avoiding some of the negative physiological consequences of hypoxia is sand training ,7. Evide−1, grass: 6.5 mmol · L−1) throughout the training session ) in highly trained runners following 21-day high-altitude, sea-level/sand, and traditional sea-level training cycles. We hypothesized that high-altitude and sea-level training on sand will significantly increase blood parameters and physical performance in comparison to the sea-level training, while a slightly greater improvement will be noted after high-altitude training.Given that the energy consumption of sand running is higher compared to training on traditional training surfaces, such as grass, this may lead to greater physiological adaptation over a specific training period. Therefore, we sought to compare differences in haemoglobin (Hb), erythropoietin (EPO) concentrations and running performance , who specialised in middle-distance and long-distance events, mountain running, with 5000 m 14:55.00 ±0:30” at the Romanian National Championships and at International Championships. The main inclusion criteria were: a compete in the national-level event and that the participants were free from musculoskeletal injuries for at least 6 months before each training cycle of the study. The athletes went through the same training program for 21 days across three training cycles separated by one year. Standard meals were provided to the athletes at each training camp. Additionally, all athletes adhered to the same regimen of vitamin and mineral supplementation. Anthropometric measures, blood draws, and running performance tests were taken on the day before and the first day after each 21-day training program to determine changes in body weight, haemoglobin concentration, and running performance. Blood samples were taken following a 12 h fast at the same time of day (±1 h) at each timepoint. Samples were stored for <48 h at 2–8 °C prior to analyses. A timeline of assessments taken before and after each 21-day training program is provided in The athletes covered three training periods, as follows: In the first stage, the athletes carried out a training cycle for 21 days at Piatra Arsă, at an altitude of ~2000 m. In the second stage, the same group of athletes carried out the same training routine at the seaside, on the Black Sea coast, at Mamaia-Constanța (~0 m). Finally, the third training cycle took place in Blaj, at an altitude of ~600 m.1 August 2017–22 August 2017, National Sports Complex “Piatra Arsă” of the Bucegi Mountains 1 August 2018–22 August 2018, on the Black Sea coast, in Mamaia-Constanța 1 August 2019–22 August 2019, in Blaj, the Alba County The timing and geographical locations of each training cycle were:A sample training program from the “Piatra Arsă” cycle is detailed in The bodyweight of participants was determined in the morning, using the same digital scale  and recorded to the nearest 100 g. Hb was determined from venous blood collected before and after each training program using a Diagon D-Cell 60 automated haematology analyser .The serum concentration of EPO was determined by the commercially available Human EPO Quantikine™ IVD ELISA Kit .2max because all subjects were over 18 years of age [The five-minute test consisted of the athletes running at full capacity for five minutes. VMA = distance obtained [in km] × 12. The five-minute test was used to determine VMA and the formula VMA × 3.5 was used to predict VOs of age . The preChamoux et al., 1996  mention 2max) remained similar prior to each 21-day training camp. Changes in these characteristics across each training camp were detected using one-way ANOVA. Repeated measures ANOVA were further used to determine differences in the relative change (% difference from pre-training) in characteristics across training camps. A Bonferroni correction was used for multiple pairwise comparisons and when sphericity was violated, the Greenhouse–Geisser corrected was used. Normality was assessed using Shapiro–Wilk’s test. Statistical significance was set a priori at p < 0.05. Statistical analyses were performed using SPSS software . Repeated measures ANOVA was used to determine whether characteristics . Given the study 2-way analysis of variance (ANOVA) (three conditions and two repeated measures), a moderate overall effect size (ES) = 0.58, an alpha-error < 0.05, the desired power (1-ß error) = 0.8, and correlation among repeated measures = 0.5, the total sample size resulted in nine participants. This value of effect size was chosen according to the improvements in VOe et al. .p = 0.133). Body weight decreased by 1.3 ± 0.6 kg (p < 0.001), from 57.7 ± 8.2 kg before the training period to 56.4 ± 8.0 kg after HIGH. There was no change in body weight from before the training period to after SAND  or from before the training period to after CTRL . The body weight of the athletes was similar prior to each 21-day training camp  in the percentage of body weight change from pre- to post-training between training cycles. Athletes lost 2.1 ± 0.4% (p = 0.001) more weight following HIGH vs. CTRL, while there were no differences in weight changes between HIGH vs. SAND (p = 0.742) and SAND vs. CTRL (p = 0.719). There tended to be an overall difference (2max was higher (p = 0.015) before the HIGH (66.7 ± 0.6 mL/kg/min) vs. CTRL (63.3 ± 0.8 mL/kg/min) training period. VO2max before SAND (65.4 ± 0.6 mL/kg/min) was not different from values before HIGH (p = 0.142) or CTRL (p = 0.231). Similarly, MAV was higher (p = 0.015) before HIGH (19.06 ± 0.18 km/h) vs. CTRL (18.10 ± 0.23 km/h). MAV before SAND (18.70 ± 0.18 km/h) was also not different from HIGH (p = 0.096) or CTRL (p = 0.135).VO2max increased by 1.7 ± 0.4 mL/kg/min following G1 (p < 0.001) and by 1.0 ± 0.2 mL/kg/min following G2 (p < 0.001). VMA increased by 0.48 ± 0.10 km/h following G1 (p < 0.001) and by 0.24 km/h following G2 (p < 0.001). However, training during G3 did not elicit a change in VO2max (p = 0.146) or VMA (p = 0.452).VO2max  and VMA . Training during G1 elicited a 2.4 ± 0.2% greater increase in VO2max (p = 0.005) and VMA (p < 0.001) compared with G3. Training during G1 also elicited a 1.0 ± 0.2% greater increase in VO2max (p < 0.001) and a 1.2 ± 0.2% greater increase in VMA (p = 0.001) compared with G2. Increases in VO2max following G2 were 1.3 ± 0.1% greater (p < 0.001) and in VMA were 1.2 ± 0.1 (p < 0.001) than those observed after G3. There was an overall effect of training cycle on the relative change in VOp = 0.003) with lower values noted before the start of G1 (5.5 ± 2.1 mU/mL) compared to G2  and G3 . Increases in EPO were elicited only with G1  and G2  training cycles. Whereas EPO were unchanged after G3 . EPO values differed prior to the start of each training camp . EPO increased to a greater extent following G1 (25.3 ± 2.7%) compared with G2  and G3 . Further, the relative increase in EPO was greater (p < 0.01) following G2 compared with G3. Training cycle had a significant effect on the relative change in EPO (p = 0.145). Each training camp provoked increases in Hb with a 0.8 ± 0.4 g/dL increase observed after G1 , a 0.5 ± 0.4 g/dL increase after G2 , and a 0.1 ± 0 g/dL increase after G3 (p = 0.003). Training cycle had a significant effect on the relative change in Hb . G1 elicited a 4.9 ± 0.9% greater increase (p = 0.001) in Hb compared with G3. G2 elicited a 3.3 ± 1.1% greater increase (p = 0.035) in Hb compared with G3. There was no difference in Hb changes observed between G1 and G2 (p = 1.0). Hb measured before the start of each training camp were similar (2max) following 21-day high-altitude, on the sand at sea level, and traditional sea-level training cycle. Our primary findings are that both, high-altitude training and sea-level training on sand resulted in significant improvements in EPO, Hb, VMA, and VO2max that exceeded changes in such parameters following traditional sea-level training. While high-altitude training elicited greater relative increases in EPO, VMA, and VO2max, sand training resulted in comparable increases in Hb and may prevent hypoxia-induced weight loss. The main conclusions of this study confirmed our initial hypothesis and suggest that the physiological modifications triggered by 21-days of either high-altitude training or training on the sand at sea level are greater than those attained by traditional sea-level training methods.This study aimed to evaluate the changes in EPO, Hb concentrations and running performance (VMA and VO2 > 0.872 L·min−1) compared to a hard surface, thus clearly demonstrating a higher level of physiological effects experiences while exercising on sand compared to traditional surfaces [The effects of altitude training have been widely examined ,24,25,26surfaces . Moreovesurfaces . Exercissurfaces  or compesurfaces . Foot prsurfaces . Furthersurfaces ,15 negatsurfaces  present surfaces  and physsurfaces .2 environment [2 carrying capacity with chronic exposure to hypoxic environments with altitude training practices have been cited to contribute to improved submaximal and maximal exercise capacity [2max values by 4.1%. Comparing this study with the one we conducted, we see similar changes. At 2000 m altitude, after 21 days of preparation, haemoglobin increased by 4.9% and VO2max values by 2.4%.The numerous studies on the effects of training in hypoxia show that one of the most important effects of altitude training concerns the increase in haemoglobin mass ,36,37,38ironment . As suchcapacity . For exacapacity . Wehrlincapacity  have shocapacity  have stu2max.Although most studies indicate favourable physiological and physical changes in increasing athletes’ performance, altitude training also has negative effects such as a decrease in body weight (in the present study by 2.1%) ,42,43,44Considering the training studies carried out thus far, additional research is necessary to determine a whole array of benefits associated with training on the sand. Namely, the main characteristic associated with exercising on sand is higher movement energy cost ,15 and tOur study is not without limitations. Although the study was conducted on elite runners, the sample size was small. Moreover, we have measured Hb concentration rather than Hb mass. Furthermore, the EPO concentrations were significantly lower prior to the high-altitude training compared with other training—this could have possibly led to the greater increases seen in this parameter following training. In addition, the differences in weather—the higher ambient temperatures on the seaside—could have led to changes in Hb mass (w/heat training).2max, while sand training resulted in comparable increases in Hb and may prevent hypoxia-induced weight loss. These findings are of practical value as they suggest that training on sand at sea-level can be considered as an alternate for high-altitude training. In this regard, when the introduction of high-altitude training is impossible, the coaches and athletes could incorporate training on sand at sea-level.The results of this study suggest that the physiological modifications triggered by 21-days of either high-altitude training or training on sand at sea-level are greater than those attained by traditional sea-level training methods. However, it must be mentioned that high-altitude training elicited greater relative increases in EPO, VMA, and VO


Escherichia coli. Reverse hydrolysis of glucose by Blg163 at pH 7.0 and 40 °C achieved a gentiooligosaccharide yield of 43.02 ± 3.20 g·L−1 at a conversion rate of 5.38 ± 0.40%. Transglycosylation of mixed substrates, glucose and cellobiose, by Blg163 consumed 21.6 U/0.5 g glucose/g cellobiose, achieving a gentiooligosaccharide yield of 70.34 ± 2.20 g·L−1 at a conversion rate of 15.63%, which is close to the highest yield reported in previous findings. Blg163-mediated synthesis of gentiooligosaccharides is the mildest reaction and the lowest β-glucosidase consumption reported to date.β-glucosidases can produce gentiooligosaccharides that are lucrative and promising for the prebiotic and alternative food industries. However, the commercial production of gentiooligosaccharides using β-glucosidase is challenging, as this process is limited by the need for high thermal energy and increasing demand for the enzyme. Here, a putative β-glucosidase gene, selected from the coral microbial metagenome, was expressed in  Bifidobacteria and Lactobacillus as probiotics, enhance vitamin synthesis, improve metabolism, inhibit the proliferation of harmful bacteria and tumors, and promote host immune response and nutrient absorption [The major component of gentiooligosaccharides is gentiobiose along with small amounts of gentiotriose and gentiotetraose, which are new functional oligosaccharides consisting of two or more D-glucose linked through β-1,6-glycosidic bonds . The strsorption ,4,5. OneExtraction from raw materials and enzymatic conversion are two main methods to prepare gentiooligosaccharides. The extraction method yields gentiooligosaccharides from the roots or stems of gentian, and bitter almond benzene. It can also be purified from the by-products of acid-hydrolyzed starch. However, these methods are complex and inefficient, and are limited by the availability of raw materials, which is difficult to meet the high demands of industrial production. The enzymatic conversion method involves the synthesis of oligosaccharides using glucose or/and cellobiose as the substrate by the reverse hydrolysis or transglycosylation of β-glucosidase. Notably, this conversion method offers the advantages of simple operation, reduced reaction steps, easy separation and purification, and high yields . HoweverAspergillus oryzae achieved a yield of 30.86 g·L−1 with cellobiose and glucose as the substrate within 72 h at pH 5 and 55 °C [Escherichia coli [and Pichia pastoris [Pichia pastoris to synthesize gentiooligosaccharides with reverse hydrolysis, as well as transglycosylation [To date, research on gentiooligosaccharide production by the enzymatic conversion method has mainly concentrated on the reverse hydrolysis of β-glucosidase that requires high levels of glucose as the raw material. β-glucosidase also displays transglycosylation activity, which is capable of synthesis of gentiooligosaccharides via the cleaving of β-1,4-glycosidic bonds of cellobiose and the transfer of substrates to glucose for β-1,6-glycosidic bond formation ,9,10. A nd 55 °C . In othehia coli  and Pichpastoris , were usEscherichia coli, or fungi, such as Aspergillus niger and yeast [Photobacterium mandapamensis, which are modestly induced on coral mucus and are antagonistic to the regulation of glucose, galactose mannose, arabinose, and N-acetyl-glucosamine, relating to the defensive capability of the coral host [β-glucosidases EC 3.2.1.21) are a typical member of the cellulases that catalyze the hydrolysis of the glycosidic bonds in beta-D-glucosides and various oligosaccharides, including disaccharides, aryl-β-D-glucosides, cyanogenic glucosides, alkyl-β-D-glucosides, and other short-chain oligosaccharides, to release glucose . A numbend yeast ,16. Thernd yeast  and zooxnd yeast ,19. Moreral host . In our  are a tyblg163, was selected from the metagenomic library of coral microorganisms using high-throughput sequencing and functional screening. After successful identification and isolation, cloning and overexpression of this gene was performed to obtain the recombinant β-glucosidase, Blg163. The recombinant protein was then characterized and assessed for its application in the industrial production of gentiooligosaccharides.In the present study, a novel gene, Escherichia coli BL21(DE3) pLysS  as the vector. T4 DNA ligase, E. coli Trans5α, restriction endonucleases, TransFast® Taq DNA polymerase and isopropyl-β-D-1-thiogalactopyranoside (IPTG) were obtained from Transgen . p-Nitrophenyl-β-D-glucopyranoside (pNPG), gentiobiose, and cellobiose were obtained from Sigma-Aldrich . N-propanol, ethyl acetate and ammonia solution were obtained from Sangon Biotech . The DNA extraction kit was purchased from Mega . Other chemical reagents used in this study were all analytically pure unless otherwise requested. Luria-Broth comprised yeast extract (5.0 g·L−1), tryptone (10.0 g·L−1), and NaCl (10.0 g·L−1), and the pH was maintained at 7.0, with or without supplementation with agar (15.0 g·L−1).pEASY-E1(+)  was used to express the recombinant protein in Coral samples collection (10 cm × 10 cm) was performed at the Luhuitou coral reef , located in the west of Luhuitou Peninsula, which is situated on Sanya Bay of the South China Sea, in September 2017. The surfaces of coral samples were washed gently with sterile seawater and transported immediately to the laboratory off-shore for isolation of β-glucosidase-producing bacteria according to Bruce Eberhart’s protocol  and our blg163 was selected and amplified using the primer pair of 5′-GCATGGAATTCTGGTGACAAAAGACATTAAAGCTCTTATTTCTCAGATGA-3′ (EcoR I digestion site underlined) and 5′-CCTCCGCTCGAGTTTCACAACAGGATTCTTCTGTACCTCATTTAACT-3′ (Xho I digestion site underlined). Then, the PCR product was cloned in the doubly digested pEASY-E1(+) empty vector to generate the recombinant plasmid pEASY-blg163, which was transformed into E. coli DH5α. The purified recombinant plasmids were subsequently transformed into E. coli BL21(DE3) pLysS for target protein expression. The transformed cells carrying pEASY-blg163 were inoculated in LB medium supplemented with ampicillin (100 µg/mL) at 37 °C. When the OD600 of the cultured bacterial cells reached 0.5~0.6, 0.6 mM of IPTG was added for further induction at 22 °C. Then, induced cells were harvested for 16 h, washed twice with phosphate buffered saline , and lysed by ultrasonication  on ice for 25 min. The lysate was centrifuged at 8200× g and 4 °C for 20 min. The supernatant was loaded onto a Ni2+-NTA agarose gel column for enzyme purification. Elution buffer  was used to elute the protein and then was diluted three times with 4 °C deionized double-distilled H2O. The purified protein was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Determination of the protein concentration was according to the Bradford method with bovine serum albumin as the standard [Based on gene annotation, the β-glucosidase gene standard .pNPG as the substrate according to previous report [pNPG, and 175 μL of 0.2 M citrate buffer (pH 7.0). After incubation at 30 °C for 5 min, the reaction was terminated by 0.1 M Na2CO3. The amount of pNP released was determined by measuring the absorption of the reaction mixture at 405 nm by spectrometer . A sample which was added 100 μL of 1 M Na2CO3 before the addition of the enzyme was set as control. The amount of enzyme that hydrolyzes 1 μmol of pNPG per min under the experimental conditions described above was defined as one unit of β-glucosidase activity (U) [Km and Vmax, pNPG, cellobiose, or gentiandisaccharide were prepared in a range of concentrations (0.25–2.5 mM) in citrate buffer (pH 7.0) and were incubated with the enzyme at 30 °C for 5 min [The enzyme activity of the recombinant β-glucosidase was determined using s report . In brievity (U) . To deteor 5 min . The conpNPG as the substrate. Synthesis of gentiooligosaccharides was detected using glucose and cellobiose as substrates. The optimal pH for the enzymatic reaction was determined using buffers with different pH from 3.0 to 11.0 at 30 °C . The optimum temperature for enzymatic reaction was determined to be from 0 °C to 70 °C in a buffer with pH 7.0. The residual activity of the enzyme was measured after incubating the enzyme at different temperatures from 0 °C to 70 °C for 1 h to determine the thermal stability of recombinant Blg163. To investigate the effects of different metal cations on the enzyme activity, 1 mM and 10 mM  of Mg2+, Li+, Na+, K+, NH4+, Ca2+, Fe3+, Ba2+, Zn2+, Ni2+, Cu2+, Co2+, diamine tetra-acetic acid (EDTA), and sodium dodecyl sulfate (SDS) were added to the reaction system individually. The effect of glucose and ethanol on enzyme activity was investigated by measuring the residual activity at different final concentrations . The sample with PBS (pH 7.0) was used as the control. All enzyme activities were determined by three repeated experiments.Purified Blg163 was characterized using w/v) glucose with 40 U of enzyme. The effect of glucose concentration on the yield of gentiooligosaccharides was studied at the optimal pH and temperature with different glucose concentrations (5–50%) incubated with 40 U of the enzyme. The effect of enzyme concentration on the yield of gentiooligosaccharides with 10, 20, 30, 40, and 100 U/g of glucose or U/0.5 g cellobiose was investigated under optimal conditions.Gentiooligosaccharide synthesis was initiated in a total volume of 2 mL in an orbital shaker  at 150 rpm. To evaluate the effect of temperature and pH on yield of gentiooligosaccharides, enzymatic reactions were conducted at a pH ranging from 5.0 to 8.0 and temperatures ranging from 25 to 45 °C in 50 mM McIlvaine buffer (pH 7.0) dissolving 20%  at 30 °C. The effects of molar ratio of substrate , substrate concentration , and enzyme concentration  on the yield of gentiooligosaccharides were determined under optimal conditions. Samples were taken at different time intervals and immediately boiled for 10 min for high performance liquid chromatography (HPLC) analysis.v/v). The spots of the samples were visualized with iodine vapor [2) column  . The mobile phase for HPLC consisted of 25% MilliQ water and 75% acetonitrile with a flow rate of 1.0 mL/min. The retention time of glucose, cellobiose, gentiooligosaccharides was 10.5 ± 0.2, 16.0 ± 0.2, 19.5 ± 0.2 min, respectively, and the calibration curve concentration ranged from 1.00 to 10.00 mg/mL. The maximal enzyme conversion was defined as 100%, and the relative conversion rate for each reaction was calculated accordingly. All analyses were performed in triplicate.The synthesis of gentiooligosaccharides was carried out by transglycosylation reaction under optimal conditions, as described above. The resulting samples were analyzed using thin-layer chromatography . The mobile phase for the TLC comprised n-propanol, ammonia solution, ethyl acetate, and water ) with accession number (MT797857). The three-dimensional structure of Blg163 was predicted by homology modeling (https://swissmodel.expasy.org/ (accessed date: 28 May 2020)) [http://smart.embl-heidelberg.de/ (accessed date: 6 March 2020)). Multiple amino acid sequences of proteins were aligned using the ENDscript server (http://espript.ibcp.fr/ESPript/ESPript/ (accessed date: 9 April 2021)) [The nucleotide sequence of the β-glucosidase gene, y 2020)) . DNA andblg163, was identified. This gene was cloned into pEASY-E1(+) and expressed in Escherichia coli BL21(DE3) pLysS strain. A complete open reading frame of 1341 bp in the putative β-glucosidase gene shared the highest identity (80%) with a gene without annotation from Celeribacter ethanolicus strain TSPH2. This gene encodes a polypeptide with 447 amino acid residues that showed the highest amino acid sequence identities of 95.3%, 94.6%, 83.2%, and 74.4% with putative proteins from the genome of Rhodobacteraceae bacterium, Celeribacter sp. HF31, Rhodobacteraceae bacterium, and Pacificibacter maritimus, respectively  [Escherichia coli BL21 (DE3) pLysS.β-glucosidase expression was induced by adding IPTG (0.6 mM) to the culture when OD54.3 kDa A, which  U·mL−1) , Blg163 R. b. NVK46632.1, β-glucosidase from Rhodobacteraceae bacterium; C. sp. WP_167600635.1, β-glucosidase from Celeribacter sp. HF31; R. b. TNE64695.1, β-glucosidase from Rhodobacteraceae bacterium; P. m.WP_123791906.1, β-glucosidase from Pacificibacter maritimus. The similar and conserved amino acids of β-glucosidases were indicated with solid and black boxes, respectively.Blg163, β-glucosidase from coral microorganism in this study; Blg163 functioned at an optimal temperature of 30 °C. Moreover, the enzyme maintained its activity up to 35.2% at 10 °C. The enzyme retained >95% residual enzyme activity after incubation at 0–30 °C for 1 h , indicat−1 for pNPGlu and cellobiose  [The effect of glucose concentration on the yield of enzymatic catalysis was determined, after an appropriate amount of β-glucosidase was assimilated to the reactions under optimal pH and temperature (pH 7.0 and 40 °C), with various concentrations of glucose as the substrate. When the glucose concentration reached 30%, the conversion rate of gentiooligosaccharides gradually increased with an increase in the concentration of glucose . Howeverglucose) . This reβ-glucosidase-mediated transglycosylation using a mixed substrate of cellobiose and glucose was adapted for gentiooligosaccharide synthesis. The yield of gentiooligosaccharides first increased and then decreased with an increase in reaction temperature, peaking at 40 °C B. The op−1 when the molar ratio of glucose to cellobiose was 4:1 (30% glucose + 15% cellobiose).Steps to increase the proportion of glucose led to a slight rise in the production of gentiooligosaccharides  A. These The synthesis of gentiooligosaccharides via reverse hydrolytic reactions requires nearly twice the amount of glucose and consumes more than 20-times the amount of β-glucosidase enzyme than transglycosylation. During the transglycosylation process, gentiooligosaccharides and glucose gradually accumulate and cellobiose is gradually deprived until the equilibrium point of the reaction is reached . The posblg163, from the coral microbial metagenome which was successfully expressed in Escherichia coli BL21(DE3) pLysS. Blg163 exhibited a reasonable tolerance to glucose and ethanol, retaining >40% activity in the presence of 5–20% glucose and >50% in the presence of 15% ethanol. Using the reverse hydrolysis activity of Blg163 to synthesize gentiooligosaccharides, we showed that a peak yield of 43.02 ± 3.20 g·L−1 and a conversion rate of 5.38 ± 0.40%, with glucose as a single substrate, could be achieved at pH 7.0 and 40 °C. We also synthesized gentiooligosaccharides via β-glucosidase-catalyzed transglycosylation with a mixed substrate of glucose and cellobiose. This process consumed 21.6 U/0.5 g glucose/g cellobiose of β-glucosidase, achieving a yield of 70.34 ± 2.20 g·L−1 and a conversion rate of 15.63%. This is the mildest reaction and the lowest β-glucosidase consumption in gentiooligosaccharide synthesis from cellobiose and glucose reported to date.This study identified a novel β-glucosidase gene, There is a patent  resulting from the work reported in this manuscript.