Interferon-γ (INF-γ) release assay (IGRA) is currently the only quantitative tuberculosis infection diagnosis method. An accurate, fast, and easily handled INF-γ detection method is the key to obtaining accurate results. Herein, we report a novel paper-based electrochemiluminescence (ECL) method based on lateral flow immunosensors that combines the easy handling characteristics of immunochromatography and the high sensitivity of electrochemiluminescence to detect IFN-γ. To our knowledge this is the first INF-γ detection method that combines immunochromatography with electrochemiluminescence. The paper-based ECL-LFI test consists of a sample pad, conjugation pad (with binding antibody IFN-γ-Ab1 conjugated with ruthenium tripyridine), detection pad (with capture antibody IFN-γ-Ab2 immobilized on nanospheres), absorbent pad, and electrode for signal activation. The ECL signal is obtained by cyclic voltammetry scanning at a speed of 0.1 V/s in the detection area of the paper-based ECL-LFI test. In our experiments, the paper-based ECL-LFI test exhibited a minimum detection limit of 2.57 pg/mL within 12 min, and a broad detection range of 2.57–5,000 pg/mL, with repeatability of 8.10% and stability of 4.97%. With the advantage of high accuracy and sensitivity, easy handling, and low user training requirements, this ECL-LFI test might be used as point-of-care testing (POCT) in the IGRA for tuberculosis diagnosis.Tuberculosis (TB) poses a serious threat to human health and social development. Accurate diagnosis of  Interferon gamma (IFN-γ) is a major immunoregulatory cytokine predominantly produced by T cells and innate lymphoid cells . IFN-γ nThe IFNB-γ release assay (IGRA) method is commonly used in the diagnosis of TB infection . The traElectrochemiluminescence (ECL) sensors incorporate a combination of electrochemistry and measurement of visual luminescence, and have the advantages of being highly sensitive, non-hazardous, and inexpensive; moreover, they exhibit a linear response over a wide range . SeveralIn this report, a lateral flow immunosensor combined with ECL was developed to detect IFN-γ. Nanospheres combined with capture antibodies were adopted to improve the capture efficiency and ECL intensity. The entire testing process takes only 12mins.The ECL-LFI adopts dry chemical diagnosis, which is different from traditional wet chemical diagnosis. Moreover, there is no need to add samples many times, samples can be added at once to detect the IFN-γ concentration without other operations, which is highly desirable for new and sudden infectious diseases. The combination of paper and screen printed electrode (SPE) makes ECL-LFI portable and mass producible. Moreover, the RPEL-B POCT electrochemical luminescence detector we used is close to a notebook, and the weight is only 1 kg, which is highly conducive to application in remote areas with poor medical conditions.2 (mcbpy-O-Su-ester) (PF6)2 (Ru-NHS ester), Butyldiethanolamine (BDEA), Tripropylamine (TPA), and TWEEN-20 were purchased from Sigma–Aldrich (United States of America). Elecsys buffer was purchased from Roche (Switzerland). Nanospheres (200-nm diameter) were purchased from Biotron Biotechnology Co., Ltd. (China). Centrifugal tubes (15 mL) and 5-mL 7K MWCO Zeba™ spin desalting columns were purchased from Thermo Fisher Scientific (United States of America). A Three Screen Printed Electrodes (SPEs) were purchased from Zensor Technology (China). Glass fiber RB-45 and absorbent paper ABP-S270 were purchased from Shanghai KingBio Biotechnology (China). Whatman No. One and Whatman No. 102 filters were purchased from Whatman (United States of America). PT-0.45 μm was purchased from Cytiva (United States of America). Biodyne C was purchased from PALL (United States of America). Heterophilic antibody blocker (HBR) and dimethyl sulfoxide (DMSO) were purchased from Bio Liye Technology (China).IFN-γ-Ab1, IFN-γ-Ab2, and antigen were purchased from Guangzhou Leide Biotechnology Co., Ltd. (China). N-Hydroxy succinimide (NHS), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydro (EDC), Ru (bpy)A RPEL-B POCT electrochemical luminescence detector was purchased from Xi’an Remex Analysis Instruments (China). An HGS210 high-speed cutting machine and HGS510-1 bench-type gold spraying marking machine were purchased from Hangzhou AutoKun Technology (China). A rotary disc mixer was purchased from Aquapro International Co. (United States of America).The method of labeling the antibody with Ru-NHS ester was based on a previously reported method with some modifications . First, Preparation of captured antibodies IFN-γ-Ab2 coupled nanospheres was performed as shown in Diluted capture antibodies was added to activated microspheres and then blocked with 0.05-M Tris-HCl and 1% Bovine albumin (BSA) before being resuspended in a resuspend solution.To determine the paper suitable for detection, different papers were cut into several discs with a diameter of 6 mm. Each disc was placed in the circular detection pad of the three-electrode SPE. Labeled antibodies was diluted to 0.2 mg/mL according to the method described in The captured antibodies was sprayed on the detection pad using the HGS510-1 bench-type gold spraying marking machine. Following that, a 2% sucrose solution was added to protect the antibody adsorbed on the paper.Each sample pad was pretreated with 0.05 M Tris (pH 7.4), 1% BSA, 0.5% sodium casein, 0.5% trehalose, 2% sucrose, 0.5 mg/mL HBR, and 0.9% NaCl. Following overnight drying in an oven at 37°C, the labeled antibodies was sprayed on the bonding pad with the HGS510-1, followed by oven drying again. The labeled antibodies was sprayed onto the conjugation pad using the HGS510-1 machine and then oven-dried again.The entire paper-based channel is 7 cm long and 6 mm wide. The sample pad is 2.5 cm long. The conjugation pad is 1.0 cm long. The detection pad is 1.5 cm long, and the absorbent pad is 2 cm long .The immunoassay was based on the double-antibody sandwich method. To begin, the sample was diluted with 0.1-M PBS (pH 7.4) and 50 μL Elecsys buffer to a final volume of 150 μL. The diluted sample was then dropped into the loading hole of the paper-based ECL-LFI. Through optimization of reaction time, following a reaction in the dark for 12 min, ECL detection was carried out as shown in t-test or analysis of variance (ANOVA) test with Bonferonni correction was applied. A p-value less than 0.05 was considered significant . All statistical analyses were performed using SPSS 21.0 software .Standard deviations were determined by relative expression ratios of three replicates for each measurement. In the case of a normal distribution, a antibody absorption×dilution factor/13700 M−1cm−1×Cantibody concentration .RPEL-B was used to measure the effect of IFN-γ-Ab2 coupled to nanospheres. The average ECL intensity of IFN-γ-Ab2 coupled with nanospheres and IFN-γ-Ab2 was 2,264 and 1,184, respectively (n = 3). p < 0.01).The ECL intensity of the materials used in the ECL-LFI test was detected to eliminate the signal interference to the Ru-NHS ester. The average ECL intensity of Ru-NHS ester, IFN-γ, IFN-γ-Ab1, IFN-γ-Ab2, nanospheres, and Elecsys buffer are 4,158.7, 79.3, 62.3, 79.8, and 113.3, respectively (n = 3). In this study we optimized several crucial parameters of the reaction of co-reactants with Ru-NHS ester. The experiment was carried out on Whatman No. 1 filter medium, and the blank group was 6 µL of 0.2 mg/mL labeled antibodies.p < 0.01).p < 0.01).p < 0.05).p < 0.01).The ECL performance for Whatman No. One and Whatman No. 102 filter media, Biodyne C, and PT-0.45 μm was tested. A 6-μL mixture of Elecsys buffer and 0.2 mg/mL labeled antibodies was dropped on each paper, and the ECL intensity was detected. The results in p < 0.01).Once the sample pad was treated by sample pad buffer, the treated sample pads were assembled in the paper-based channels with detection pads made of Whatman No. 1, Whatman No. 102, and Biodyne C, and the ECL intensity was measured under the same conditions for each paper. The sample pad buffer can increase the hydrophilicity of the paper and the pretreatment of samples, balance the pH of the sample to be tested, and adjust the salt ion strength. The results in p < 0.01). No significant ECL intensity was found in the 0.7-cm group. The reason is that the detection pad should cover the electrodes completely, and it also should overlap the front and rear combination pads and water absorption pad by a small amount. Since the 0.7-cm detection pad length is too short, the normal flow measurement and chromatography process cannot be completed.p < 0.01). Considering the cost of the paper-based ECL-LFI test, no more experiments were conducted on other concentrations.To obtain higher ECL intensity, we set four groups of labeled antibodies to capture antibodies concentration ratios as follows for the ECL intensity test: 1 mg/mL:0.25 mg/mL, 1 mg/mL:0.5 mg/mL, 1 mg/mL:1 mg/mL, and 1 mg/mL:2 mg/mL. All were joined to 150 μL 1,000 pg/mL samples. R2 = 0.9932. Moreover, the detection limit for IFN-γ was 2.57 pg/mL (S/N = 3) according to the Guidelines of the Clinical and Laboratory Standards Institute (NCCLS)  . The resThe reliability of the paper-based ECL-LFI test was verified by using a commercial ELISA test. Upon adding serum of unknown concentration, ECL-LFI found a result of 206.5 ± 17.6 pg/mL; the ELISA result was 201.3 ± 10.2 pg/mL. p < 0.01).The as-prepared ECL-LFI test was investigated to evaluate its specificity, reproducibility, and stability. Ten paper-based ECL-LFI tests were used to detect 1,000 pg/mL IFN-γ. RSD for IFN-γ is 8.1%, indicating an acceptable reproducibility of the paper-based ECL-LFI . The staThe paper-based ECL-LFI test we developed enabled us to use a linear channel for electrochemical detection of IFN-γ in human serum, for the first time. This development enables us to add samples at one time and detect IFN-γ concentration in human serum without other operations. The proposed paper-based ECL-LFI test provides highly sensitive ECL signals. Moreover, the commercial ELISA test results demonstrate that the paper-based ECL-LFI test is highly reliable. Therefore, the paper-based ECL-LFI provides an effective approach for detection of IFN-γ in human serum and has potential application in facilitating accurate and reliable clinical diagnosis of TB. In addition, the successful preparation of the proposed ECL-LFI test can be applied to the detection of other diseases and lays a foundation for the preparation of multiple ECL paper-based ECL-LFI tests with multiple channels and multiple detection projects in the future.


This study revealed key differences related to the mechanism utilized by M and T to increase contractile force generation and suggests that targeting different proteins within the sarcomere may result in differentiating therapeutic profiles. Sarcomeres, the basic functional unit of striated and cardiac muscle, facilitate muscle contraction through the movement of the actin thin filament over the myosin thick filament. This process is coupled to myosin ATPase activity and regulated both by changes in intracellular calcium concentrations and by post-translational modifications to the sarcomere proteins.11,Omecamtiv mecarbil (OM) improves contractile performance by stimulating myosin.,,,,Several post-translational modifications to the sarcomere resulting in abnormal contractile performance are associated with HF.,,AAA and displayed no changes in morbidity or mortality, but displayed depressed cardiac contractility, altered sarcomere structure, and upregulation of transcripts associated with a hypertrophic response.,,,,,,,,Here, we used a transgenic mouse model in which phosphorylation sites at serines 273, 282, and 302 within the m-domain of cMyBP-C were replaced with the phospho-null residue alanine (AAA).The study aims were to compare the effects of a myosin selective small molecule that is an OM surrogate, CK-1317138 (CK-138 or M), with a troponin selective small molecule, CK-3826030 (CK-030 or T), on contractile mechanics, as well as to further investigate the impact that cMyBP-C phosphorylation has on their activity. To answer these questions, a series of experiments with M and T were performed using cardiac tissue and cells isolated from all 3 transgenic lines. The study demonstrated that both myosin- and troponin-selective compounds were able to improve contractile function irrespective of the cMyBP-C phosphorylation state. This study additionally revealed differences between M and T, where T leads to greater force redevelopment and ATP exchange rates.The transgenic mice used for the experiments were derived from wild-type mice in the FvB/N background. The 3 serine residues  in the m-domain were replaced by alanine residues for the AAA cMyBP-C hypo-phospho-mimetic transgenic ,ktr) was measured (Aurora Scientific Inc) as previously described.ktr was calculated by fitting the force redeveloped in the fiber, after this slack-stretch treatment against the time required to one-phase association curve.Papillary fibers were carefully dissected from the left ventricles of 12-14 weeks old FVB/N mice of all 3 genotypes .,2, 8 mmol/L; EGTA, 5 mmol/L; K2HPO4, 5 mmol/L; KH2PO4, 5 mmol/L; NaN3, 3 mmol/L; ATP, 5 mmol/L; DTT, 1 mmol/L; BDM, 20 mmol/L; Triton-X 100, 0.1%, pH 7.0) for 6 hours at 4°C. The skinning buffer was changed every 2 hours. After 6 hours, skinned fibers were then immersed in glycerinating buffer  overnight at 4°C under gentle agitation. For the mANT-ATP turnover experiments, a small, glycerinated fiber (∼800 μm × 150 μm) was placed on a cooled slide. The fiber was fixed onto the slide by double-sided tape to create a chamber using a glass cover slip. The glycerinated fiber was washed with rigor buffer  5 times, followed by a final wash by rigor buffer with 250 μmol/L of mANT-ATP. The fibers were incubated in the presence of mANT-ATP for 5 minutes. Later, the slide was placed in the microscope chamber, and the 10× objective of Leica DMi8 microscope was used to image the slide. The slide was imaged every 5 seconds for 600 seconds by exciting the slide at 395 nmol/L by DAPI filter cube with exposure of 20 milliseconds to avoid photobleaching. After 60 seconds, the mANT-ATP buffer was washed away with ATP-chase buffer  to allow the myosin in the fiber to exchange the fluorescent mANT-ATP with regular nonfluorescent ATP. For M and T, the glycerinated fibers were treated with the rigor buffer, mANT-ATP buffer and ATP-chase buffer containing the indicated concentration of the compound.The nucleotide chase experiments were performed as described previously.F and TS = time constants for the lifetime of the 2 phase mANT-ATP exchange.The decay in fluorescence was calculated by subtracting the background fluorescence from the fluorescence of the fiber at 3 different regions of the fiber. The decay in fluorescence was fitted to a custom defined 2-state exponential decay curve to yield the biphasic exchange of mANT-ATP with ATP in accordance with decrease in fluorescence:2, 5 HEPES, and 11 D-glucose, pH 7.4) at 2.5 ml/min, 37°C for 4 minutes. The heart was then perfused with enzyme solution using the adult mouse cardiomyocyte isolation kit following the manufacturer’s instructions . After digestion, the heart was removed from the cannula and the left ventricle was gently teased apart. Cell suspensions were obtained by gentle pipetting. After spinning, cardiomyocytes were transferred to Tyrode buffer with Ca2+.Left ventricular cardiomyocytes were isolated from cMyBP-C AAA, cMyBP-C DDD, and the control cMyBP-C transgenic tWt mice. Following heart excision, the aorta was cannulated on a Langendorff apparatus (Harvard Apparatus). The heart was perfused retrograde with Tyrode buffer . After stabilization, the myocytes were perfused with perfusion buffer at room temperature. They were field stimulated with 12 V at 1.0 Hz with a 1.0 milliseconds pulse width using a pair of platinum wires placed on opposite sides of the chamber connected to a MyoPacer stimulator. Myocytes perfused with perfusion buffer were recorded at baseline, then perfused with compound for 3-4 minutes. Analysis was performed using the IonWizard software (IonOptix). For each test, an average of 10-15 sarcomere shortening traces was analyzed. Cells were selected that were beating and appeared grossly normal. Cells were not considered for recording if they were not contracting or, in very rare instances, appeared to be exhibiting an abnormally large reduction in sarcomere length . All recorded cells were analyzed. Cells with poor data quality were excluded. The remaining cells were recorded and exhibited a 7%-15% reduction in sarcomere length relative to its resting sarcomere length at baseline before treatments were given. Cells were isolated from 2 to 4 animals for each group. Neither the genotype nor the treatment disproportionately influenced cell exclusion.Cardiomyocyte contractility was measured using an IonOptix contractility system. Myocytes were loaded in the perfusion chamber of the microscope stage filled with perfusion buffer  and then homogenized with 1300D Polytron Homogenizer (12-mm generator) for 2 minutes at 30,000 revolutions/min on ice. The homogenization step was repeated 3 times. The homogenate was centrifuged at 5,000 revolutions/min for 10-15 minutes. After centrifugation, all the supernatants were discarded, and a light brown layer of mitochondria, sarcolemma, and sarcoplasmic reticulum on top of the pellet was scraped off. The pellet was homogenized with buffer B . The suspension was then centrifuged at 9,000 rpm for 15 minutes. The supernatant was discarded, and a light brown layer on top of the pellet was scraped off. The pellet was homogenized with buffer C containing 1% Triton X-100  to remove mitochondrial, sarcolemmal, and sarcoplasmic reticular membranes. After the pellet was fully resuspended in buffer C, the suspension was then centrifuged at 4,200 revolutions/min for 10 minutes. This sequence of resuspension, homogenization, and centrifugation was repeated another 2 times. The resulting light brown pellet was filtered through Nitex nylon mesh, 630 MICRON , to remove any residue of coarse tissue. Then, the previous sequence of resuspension, homogenization, and centrifugation was repeated another 2 times. Finally, Triton X-100 was removed with buffer A wash 3 times. In each wash, the pellet was homogenized in Buffer A, and the suspension was centrifuged at 4,200 revolutions/min for 10 minutes. All supernatants were discarded, and the top light brown layer of each pellet was removed. After 3 washes, the pellet was homogenized in buffer A. The homogenate obtained after 3 washes was white in color. The homogenate was then aliquoted and stored at −80°C.Mouse myofibril preparation: 35 to 50 frozen mouse hearts were defrosted on ice. The tissue was chopped in a Waring blender with cold buffer A  over 7 points, and the concentration of compound in the last point was 0. The compound solution at each concentration was dispensed at 0.50 μL into the plate. Myofibrils were fully thawed on ice and homogenized with 1300D Polytron Homogenizer for 30 seconds at 20,000 revolutions/min. Then, the homogenized myofibrils were diluted to the desired concentration and 10 μL myofibril solution was dispensed into the plate. A reaction solution containing 4.0 mmol/L ATP, 1.6 mmol/L NADH, 3.0 mmol/L phosphoenolpyruvate, 30 U/mL lactate dehydrogenase, and 30 U/mL pyruvate kinase was freshly prepared and dispensed at 25 μL into the plate to initiate the myofibril-catalyzed reaction. Upon finishing dispensing, the 384-well plate was kinetically read at 340 nm at every 30 seconds for 1 hour.The experiment was performed in an assay buffer containing 15 mmol/L PIPES (pH 7.0), 5.0 mmol/L MgCl2, and 1 mmol/L DTT, pH 6.8. The steady-state ATPase activity in these reconstituted sarcomeres was analyzed by the oxidation of NADH to NAD+ to convert the ADP produced by active myosin S1 heads to ATP aided by pyruvate kinase and lactate dehydrogenase. The absorbance for NADH was measured at 340 nm in 25°C using a SpectraMax plate reader (Molecular Devices).The myosin ATPase activity in a reconstituted sarcomere were performed based on the previous study.P > 0.05, then we assumed a normal or parametric distribution. Statistical analyses for significance were performed using GraphPad Prism software (version 9.3) using either a 1- or 2-way analysis of variance (ANOVA), as applicable. For parametric distributions, ANOVA analyses used either Sidak's (comparing to a control) or Tukey’s  post hoc method for multiple pairwise comparisons. For nonparametric distributions, ANOVA analysis used a Kruskal-Wallis test post hoc method for multiple pairwise comparisons. A repeated measures ANOVA was used for within group 2-way ANOVA comparisons. A P value <0.05 was considered statistically significant. Data are represented as the group mean ± SEM.A Shapiro-Wilk test was used to test for normality using the scipy python library (version 1.6.2) within JupyterLab Note-book (version 6.3.0) distributed through Anaconda (version 3). If Both compounds activate the sarcomere to produce more contractile force. The rate of ATP consumed by the sarcomere during cross-bridge cycling is an indirect assessment of contractile force that can be measured by monitoring steady-state ATPase activity from myofibrils isolated from cardiac tissue. M (CK-138) and T (CK-030) selectivity were evaluated using a reconstituted hybrid sarcomere protein ATPase assay that was previously used to determine OM selectivity and, more recently, selectivity for CK-136, a small molecule that selectively alters troponin activity to increase cardiac muscle contraction.The reconstituted sarcomere consisted of myosin S1 and thin filaments from either cardiac, fast, or slow skeletal muscle. Selectivity can be measured, because the molecules exhibit differential activity depending on the source of myosin and troponin. Here, we reconstituted an all-cardiac and an all-skeletal sarcomere as well as hybrid systems where myosin comes from cardiac but troponin comes from skeletal and vice versa. For M, we saw activity that tracked with the source of myosin. The reconstituted sarcomere with cardiac muscle myosin S1 had increased steady-state myosin ATPase activity in presence of 40 μmol/L M irrespective of thin filament origin . M is a −4 AU/min, compared with a rate of 0.023 ± 7 × 10−4 AU/min in the presence of compound T  produced by AAA papillary fibers (22.6 ± 1.9 and 23.4 ± 2.3 mN/mm2) at pCa 4.5 was significantly attenuated relative to tWt (41.9 ± 2.7 and 42.7 ± 2.4 mN/mm2) and DDD fibers (39.6 ± 3.0 and 38.1 ± 3.0 mN/mm2) (solid lines in max in tWt (47.7 ± 3.1 and 46.3 ± 3.8 mN/mm2), AAA (27.0 ± 3.4 and 26.6 ± 3.2 mN/mm2), and DDD (46.6 ± 3.0 and 49.8 ± 9.4 mN/mm2), except at the 3× dose in fibers isolated from AAA transgenic mice and the 1× dose in fibers isolated from DDD transgenic mice (max for papillary fibers isolated from tWt (47.1 ± 2.6 and 54.7 ± 4.6 mN/mm2) and AAA (26.5 ± 2.6 and 31.2 ± 4.0 mN/mm2), whereas the 1× dose led to a significant increase in fibers isolated from DDD (41.2 ± 3.6 and 49.5 ± 8.0 mN/mm2) transgenic mice.We next performed experiments measuring force production per cross-sectional area in skinned cardiac papillary fibers isolated from tWt, AAA, and DDD transgenic mice. The force produced in skinned papillary fibers was measured at 1× and 3× concentrations of M and T. Consistent with the myofibril ATPase activity assay results, maximal force  of the fibers. For papillary fibers isolated from tWt (5.69 ± 0.01 and 5.68 ± 0.01), AAA (5.69 ± 0.01 and 5.7 ± 0.01), and DDD (5.64 ± 0.01 and 5.65 ± 0.01) transgenic lines, there was no significant difference found between the pCa50 in untreated fibers , AAA (5.82 ± 0.01 and 6.00 ± 0.02), and DDD (5.77 ± 0.01 and 5.93 ± 0.01), respectively. 1× and 3× of T also increased the pCa50 in tWt (5.80 ± 0.01 and 5.89 ± 0.01), AAA (5.80 ± 0.01 and 5.93 ± 0.02), and DDD (5.75 ± 0.01 and 5.88 ± 0.01), respectively. The increase in calcium sensitivity upon compound treatment indicates an improvement in force-producing capability for skinned fiber at low calcium concentrations.We also measured the effect of M and T on the calcium sensitivity  was also measured at a submaximal calcium concentration, pCa 5.7. The ktr for DDD displayed a significantly higher ktr (5.77 ± 0.23 and 5.79 ± 0.18 s−1) relative to AAA (4.23 ± 0.15 and 4.21 ± 0.19 s−1) or tWt (4.32 ± 0.18 and 4.29 ± 0.13 s−1)  (ktr) across all genotypes   , Table 244 s−1]) , Table 287 s−1]) , Table 2max when compared with untreated . Representative traces from untreated and from M- and T-treated fibers illustrate the prolonged attachment of the myosin heads to actin, which impeded detachment leading to a significant reduction in the % drop in Fntreated . The 1× ntreated , Table 2ntreated , black. ,We next wanted to compare ATP turnover rates, because it has been reported that OM prolongs the duration of the strongly bound state.The slow phase for mANT-ATP turnover, or exchange, was comparable for fibers isolated from tWt (24.6% ± 0.8% and 23.8% ± 0.7%) and AAA (27.6% ± 1.3% and 25.6% ± 0.6%) mice but was significantly lower in fibers isolated from DDD mice (12.98% ± 0.4% and 14.1% ± 0.4%)This study evaluated 2 new small molecules that target 2 different sarcomere proteins to increase contractility. M, a myosin targeting small molecule surrogate of OM, modulates the activity of myosin heads, while T, a surrogate of CK-136, targets the troponin complex. Both compounds result in a leftward shift of the force-calcium curve, indicating an ability of the muscle to produce more force for a given calcium concentration to augment cardiac muscle contraction efficiency.,cMyBP-C phosphorylation is a common post-translational modification associated with HF where reduced phosphorylation impairs contractility by decreasing the number of myosin heads that are actively engaged in cross-bridge cycling. Given the fact that cMyBP-C is more than 50% dephosphorylated in failing hearts regardless of disease etiology,cMyBP-C phosphorylation status did not alter the effectiveness of either M or T. Nevertheless, maximal force remains attenuated in the AAA transgenic line, because reducing the level of phosphorylated cMyBP-C reduced the population of myosin heads actively undergoing cross-bridge cycling. Our data therefore indicate that both M and T increase force generation by modulating cross-bridge cycling for active myosin heads. Our work is largely consistent with recently published work that evaluated the effect of OM on force generation using myocardial tissue isolated from wild-type and phosphorylation-deficient cMyBP-C mice.ktr) and suggest that, at concentrations near their respective in vitro EC50s, T led to a greater rate of ATP exchange. The dose dependent decline in the percent drop in Fmax with M at these concentrations upon slack provides additional evidence, as the drop in Fmax is indicative of myosin head - thin filament release and suggests that M impeded this action.This study revealed key differences related to the mechanism used by M and T to increase contractile force generation. The force mechanic experiments showed that T contributed to a much faster tension redevelopment rate , the phosphorylation deficient (AAA), and wild-type cMyBP-C (tWt). Although these lines have been well characterized,Furthermore, it is worth noting that cMyBP-C is itself an activator of muscle. Recent studies have shown that the N-terminal fragment of cMyBP-C increases force in permeabilized cardiomyocytesMYH7 selective.PerspectivesCOMPETENCY IN MEDICAL KNOWLEDGE: HF is a grievous condition with poor prognosis. Current treatments that reduce HF-related death and hospitalizations are focused on antagonizing the neurohormonal and hemodynamic stress axes. Directly targeting sarcomere proteins represents a potentially new therapeutic class for HF management that directly target the basic functional unit of cardiac muscle to increase contraction efficiency.TRANSLATIONAL OUTLOOK: This work provides evidence that selectively activating different proteins within the sarcomere can increase muscle contraction by different means. We believe this could result in differentiating therapeutic profiles for this future class of medicines.Additionally, translatability is always a concern. The studies were performed primarily on mice, where MYH6 is the dominant form of cardiac myosin, whereas humans express the MYH7 variant. M is a surrogate of OM, and OM has been shown to be Dr Sadayappan has received support from National Institutes of Health grants R01 AR078001, R01 HL130356, R01 HL105826, R38 HL155775, and R01 HL143490; and provides consulting and collaborative research studies to the Leducq Foundation (CURE-PLAN), Red Saree Inc, Greater Cincinnati Tamil Sangam, Novo Nordisk, Pfizer, AavantiBio, AstraZeneca, MyoKardia, Merck, and Amgen, but such work is unrelated to the content of this work. All other authors were employed by Amgen at the time the experiments were conducted.


Laminaria digitata (Ochrophyta) in which decay was induced. Subsequent sequencing of a fosmid clone insert revealed the presence of a gene encoding a bifunctional phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme 10L6AlgC, closely related to a protein from the halophilic marine bacterium, Cobetia sp. 10L6AlgC was subsequently heterologously expressed in Escherichia coli and biochemically characterized. The enzyme was found to possess both PMM and PGM activity, which had temperature and pH optima of 45°C and 8.0, respectively; for both activities. The PMM activity had a Km of 2.229 mM and Vmax of 29.35 mM min−1 mg−1, while the PGM activity had a Km of 0.5314 mM and a Vmax of 644.7 mM min−1 mg−1. Overall characterization of the enzyme including the above parameters as well as the influence of various divalent cations on these activities revealed that 10L6AlgC has a unique biochemical profile when compared to previously characterized PMM/PGM bifunctional enzymes. Thus 10L6AlgC may find utility in enzyme-based production of biochemicals with different potential industrial applications, in which other bacterial PMM/PGMs have previously been used such as in the production of low-calorie sweeteners in the food industry.Macroalgae host diverse epiphytic bacterial communities with potential symbiotic roles including important roles influencing morphogenesis and growth of the host, nutrient exchange, and protection of the host from pathogens. Macroalgal cell wall structures, exudates, and intra-cellular environments possess numerous complex and valuable carbohydrates such as cellulose, hemi-cellulose, mannans, alginates, fucoidans, and laminarin. Bacterial colonizers of macroalgae are important carbon cyclers, acquiring nutrition from living macroalgae and also from decaying macroalgae. Seaweed epiphytic communities are a rich source of diverse carbohydrate-active enzymes which may have useful applications in industrial bioprocessing. With this in mind, we constructed a large insert fosmid clone library from the metagenome of  Laminaria digitata (Hudson) J.V. Lamouroux  is a dark-brown seaweed  which is typically distributed in European coastal waters in areas ranging from the southern coast of Brittany in France to Iceland and the north coasts of Scandinavia  of phenolics from the macroalga Fucus vesiculosus  strategies and improvements in the yields of algal components with functional food, nutraceutical, biopharmaceutical and cosmetics applications  enzyme which we subsequently sub-cloned, expressed, and biochemically characterized. PMM/PGM are members of the alpha-d-phosphohexomutase superfamily of enzymes and catalyze the reversible conversion of 1-phospho to 6-phosphohexoses, involving various different sugars such as glucosamine, glucose, mannose, and N-acetylglucosamine. Optimum enzyme activities for both PGM and PMM activities were observed at 45°C and pH 8.0. The highest PGM activity was observed with Mg2+ as a co-factor but PMM activity was highest with Mn2+.With this in mind, we targeted the microbiome of d-Glucose-1-phosphate disodium salt (G1P), α-d-Mannose-1-phosphate sodium salt (M1P), and α-d-Glucose-1, 6-diphosphate potassium salt were purchased from Biosynth Carbosynth (United Kingdom). NADP and NADPH were purchased from Sangon Biotech (China). Phosphoglucose isomerase, phosphomannose isomerase, and glucose-6-phosphate dehydrogenase were purchased from Sigma-Aldrich (United States).α-Laminaria digitata, including blade, meristem, stipe, and holdfast regions, was collected from Finavarra in Co. Clare, Ireland, at 53° 08′ 59″ N, 9° 08′ 09″ W, as previously described , to 950 ml sterile artificial seawater , in each of three 2 l flasks and the flasks were incubated at different temperatures  on a shaking platform at 125 rpm for a 6-week period. Triplicate 5 g samples were withdrawn from each flask after 2, 4, and 6 weeks. Samples were stored at −80°C until further use.escribed . All sam2PO4, 5 M NaCl, and 10% CTAB) and incubated for 2 h at 70°C. Each sample was split in half and 1 ml of chloroform: isoamyl-alcohol (24:1) was added to each in 2 ml Eppendorf tubes, mixed by vortexing, and then centrifuged at 9,000 × g. The upper phase was transferred to fresh Eppendorf tubes and 0.7 vols isopropanol and 0.1 vols 3 M sodium acetate pH 5.2 was added to each tube. Tubes were inverted several times to mix and were then centrifuged for 30 min at 25,000 × g. Supernatants were discarded and pellets were washed with cold (4°C) 70% EtOH . Ethanol was discarded and the pellets were air-dried at room temperature and then dissolved in TE buffer. Each DNA sample was treated with proteinase K and RNase A. All samples were then pooled. An equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added. Tubes were centrifuged at 25,000 × g for 30 min. The upper phase was transferred to fresh tubes and the DNA was precipitated with isopropanol and sodium acetate as before and washed with cold ethanol as before. DNA was quantified by NanoDrop ND-1000 and viewed by electrophoresis on a 1% agarose gel.Total mDNA was extracted individually from 1 to 1.5 g of each of 30 samples . Samples were thawed on ice and suspended in 1 ml of lysis buffer  at 6 V/cm, 1–25 s switch time, 120° angle, 17 h, 14°C in 0.5X TBE buffer  on a 1% PFGE grade agarose gel (Bio-Rad Laboratories). For size comparison, each gel contained a PFGE DNA Molecular marker . The marker lane and approx. 0.5 cm of the sample DNA lane were cut off and stained for 30 min in SafeView dye for visualization. A gel slice containing unstained DNA of about 30–50 Kb was excised and the DNA was electro-eluted from the gel slice and concentrated as described by BstZ17I  and dephosphorylated using shrimp alkaline phosphatase  and subsequently purified by phenol extraction after inactivating the enzymes by incubation at 70°C. A ligation reaction was performed with ~0.25 μg insert (20–50 Kb mDNA) and ~0.5 μg linearized vector T4 ligase . Ligation reaction products were packaged in Lambda Phage packaging extracts  and transfected into E. coli EPI300™ host cells. The transfection reaction products were spread to Q-Trays on LB agar including kanamycin (50 μg/ml) and chloramphenicol (12.5 μg/ml) and clones were grown by incubating at 37°C. Clones were hand-picked from isolated colonies into LB broth (including kanamycin and chloramphenicol) in 384 well microplates (Genetix) and grown by incubating at 37°C. Subsequently clones were replicated to 384 well microplates containing storage medium  for long-term storage.Fractionated HMW DNA was end-repaired using the End-It™ end repair kit (Epicentre) according to the manufacturer’s instructions and metagenomic libraries were constructed using the vector pCCERI, a fosmid derived from the commercial pCC1FOS (Epicentre Biotechnologies) vector . The cirde novo assembly with CLC Genomics Workbench (version 4.22.107090). Assembled contigs were annotated using PROKKA v.3.2.1 (Clone 10L6 was grown overnight in LB broth supplemented with kanamycin (50 μg/ml), chloramphenicol (12.5 μg/ml), and 0.1% arabinose. Cells were pelleted and the fosmid was extracted with the FosmidMax (Lucigen) extraction kit, according to the manufacturer’s instructions. Fosmid (including contaminating genomic DNA) was sequenced by Illumina HiSeq . Sequences were assembled by Eurofins Genomics Ltd., using  v.3.2.1  on the K v.3.2.1 .algC gene sequence (Accession No.: ON876002) was codon optimized for expression in E. coli and synthesized by Sangon Biotech (China). The algC was sub-cloned into the NdeI-EcoRI sites of the pMAL-c5X vector  to allow for the production of PMM/PGM containing a maltose-binding protein (MBP) tag at the N-terminus. The recombinant plasmid was subsequently transformed into competent E. coli BL21 (DE3) . Cultures were grown at 37°C until OD600 reached 0.6, and induced with 0.3 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 16 h at 16°C. The cells were harvested by centrifugation at 8,000 rpm for 10 min at 4°C, and pellets were rapidly frozen with liquid nitrogen and stored at −80°C.The 10L6Purification was performed on the MBPTrap HP 5 ml column  using ÄKTA Pure . Cell paste which was obtained following centrifugation of 500 ml of culture was resuspended in 30 ml binding buffer  and was sonicated until the solution was clear. The supernatant was obtained by centrifugation at 13,000 rpm for 40 min and purified using the MBPTrap HP column which had been equilibrated with binding buffer (pH 7.4). PMM/PGM was eluted from the column by washing with binding buffer supplemented with 20 mM maltose. The eluted protein was further purified with a HiLoad™ 16/60 Superdex™ 200 pg. column , which had been pre-equilibrated with buffer containing 20 mM Tris–HCl (pH 7.4), and 150 mM NaCl. Fractions containing PMM/PGM were identified by SDS-PAGE. The concentrations of the protein fractions were measured by UV–Vis Spectrophotometer .2, 1 mM NADP+, 0.1 mM glucose-1,6-bisphosphate, 1 mM G1P, and 0.5 U glucose-6-phosphate dehydrogenase. PMM activity was measured in a similar mixture by replacing G1P with 1 mM M1P and supplementation with 0.5 U phosphoglucose isomerase and 0.5 U phosphomannose isomerase. A NADPH standard curve for both PGM and PMM activity was generated with a series of standard solutions at concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 1.0 mM, respectively  for 10 min and then assaying the PMM/PGM activities in the standard reaction. The effect of NaCl concentration on PMM/PGM activity was measured in mixtures of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 M NaCl, respectively. All the aforementioned experiments were conducted in triplicate.The optimum temperature for activity was determined by monitoring enzyme activity at 25, 30, 35, 40, 45, 50, and 55°C in 50 mM Tris–HCl (pH 7.5), respectively. The optimum pH value was determined by measuring the reaction in mixtures at pH values of 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5 at 45°C. To study the effects of metal ions, MgClKinetic analysis was conducted by adding G1P ranging from 0 to 2 mM and M1P ranged from 0 to 4 mM M1P, respectively, at optimal reaction condition, with each reaction being conducted in quadruplicate. Kinetic parameters were fitted to the Equation S1+S/Ki  and calcThe SWISS-MODEL template library  was searched with BLAST  and HHBlProtein sequence alignments were performed with (i) T-Coffee  and (ii)E. coli chromosomal sequences, and (iv) a cloned fosmid insert sequence of 34,905 bp in length which shared 99% similarity (94% query coverage) with a Cobetia sp. (strain L2A1) complete genome sequence (accession no. CP047025).Illumina HiSeq sequencing yielded 4,235,950 reads which were assembled to 2,932 contigs, with a coverage of 290x over 4,366,821 sites. These included 67 contigs larger than 10 Kb, 76 contigs between 5 and 10 Kb in length, and 2,789 contigs smaller than 5 Kb. BLAST analyses of all contigs revealed sequences which mapped to (i) a helper plasmid sequence with 99.95% sequence identity to pRH220 (accession no.: AB526840) from the cloning host, (ii) a complete cloning vector (pCCERI) sequence (12.3 Kb) , (iii) calgC), potentially involved in carbohydrate metabolism (Cobetia sp. L2A1 (99.5% similarity). The translated protein consisted of 460 amino acid residues. The protein was similar to other PMM/PGM proteins from Cobetia spp., including Cobetia marina, and also displayed similarities to proteins from Chromohalobacter spp. and Halomonas spp., and to Chromohalobacter spp. enzymes  than PGM (43% relative activity at 0.2 M); but as NaCl concentrations increased, similar decreases in relative PGM and PMM activities were evident ,C. Finalobserved , while t for M1P . The KcaPseudomonas aeruginosa PAO1 (PDBe code: 2h4l) (d-ribofuranose (R1P)  of 100%, exceeding the threshold values in both cases. The overall PROCHECK G-score was predicted to be −0.12  and the ProSA Z-score was −8.15 (within native conformation range), with both indicating good model quality. PROCHECK Ramachandran plot calculations revealed 92.6% of amino acid residues in the most favored regions, with an additional 7% in allowed regions, and 0% in disallowed regions.10L6AlgC was modeled using the SWISS-MODEL server, based on the top template, a 463 amino acid PMM/PGM enzyme, from e: 2h4l) , with bose (R1P) . The 10LL. digitata. Previous reports had highlighted that bacteria associated with macroalgae such as A. nodosum are enriched in macroalgal-polysaccharide-degrading bacteria  is involved in the reversible interconversion of glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) and is a key enzyme linking the glycolytic and gluconeogenesis pathways in bacteria . When incharides  in many ruginosa . GDP-d-mruginosa , and GDPcomitans .algC, are key virulence factors in P. aeruginosa (Mycobacterium tuberculosis (M. tb)  resulted in almost complete loss of the O-antigen polysaccharide   and in Hr pylori . P. aerue models . M. tb irophages . PMM/PGMccharide . Thus, tccharide . PGM act for CPS . The prothe host . α-PGM hs lactis . PMM gene strain . This ine strain . EPS froactivity .Ruminiclostridium cellulolyticum, where it converts a-d-glucose-1-phosphate (Glu1P) produced by both cellobiose phosphorylase and by the Leloir pathway, into d-glucose 6-phosphate (Glu6P), which then enters the glycolytic pathway  and PMM (manB) genes in the strain resulting in enhanced bleomycin production in the strain  biosynthesis in the animal pathogen e strain .Cobetia sp. L2A1 and given the high similarity between the ~35 Kb cloned fosmid insert and the Cobetia sp. L2A1 genome sequence (99% identity), it is probable that the cloned DNA is from the same species. Cobetia sp. L2A1 was originally isolated from a brown algae and first identified in an alginate utilization screen  from the same family   . StructuE. coli (P. aeruginosa (Acetobacter xylinum (Clostridium thermocellum (Thermococcus kodakaraensis (Pyrococcus horikoshii (P. aeruginosa (50 kDa) (T. kodakaraensis (48–50 kDa) (P. horikoshii (50 kDa). It is however smaller than those from E. coli (62 kDa) (A. xylinum (59 kDa) (C. thermocellum (65 kDa)  , T. koda–50 kDa) , and P. (62 kDa) , A. xyli(59 kDa) , and C. (65 kDa) . The E. Km of the 10L6AlgC PGM activity (0.5314 mM) was greater than that of many of the aforementioned enzymes; such as E.coli (0.05 mM) (P. aeruginosa (0.022 mM) (A. xylinum (0.0026 mM) (C. thermocellum (0.41 mM) (P. horikoshii (0.09 mM) (T. kodakaraensis (3.0 mM) , but again as with the PGM activity was lower than that of T. kodakaraensis (3.2 mM).The 0.05 mM) , P. aeru.022 mM) , A. xyli0026 mM) , C. ther0.41 mM) , as well0.09 mM) . It was (3.0 mM) . SimilarE. coli (pH 9.0) (C. thermocellum (pH 8.8) (P. horikoshii (pH 9.0) (A. xylinum (pH 5.5–7.4) (T. kodakaraensis (pH 7.0).With respect to optimal pH, the optimum pH for 10L6AlgC activities was 8.0, which was slightly less alkaline than the aforementioned enzymes which ranged from pH 8.8–9.0; such as those from (pH 9.0) , C. ther(pH 8.8) , and P. (pH 9.0) . In cont5.5–7.4)  and T. k2+, Mn2+ increased the PMM activity but decreased the PGM activity. Reduced enzyme activities were also seen in the presence of Ca2+, Co2+, Li2+, Na2+, Ni2+, and Zn2+. This PMM/PGM activity profile differs from the activity profile from the aforementioned characterized enzymes in other strains. For example in E. coli Ni2+ and Zn2+ did not affect PGM activity, while Mn2+ resulted in reduced activity  (P. aeruginosa enzyme (<37°C)  ; but is  (<37°C) .Cobetia spp., Chromohalobacter spp., and Halomonas spp. (data not shown) reveal that the metal co-factor binding (DGDGD) and catalytic site (TGSHNP) motifs are strictly conserved in the family. Of the 13 amino acids interacting with the substrate and ligand, 7 of them form 10 H-bonds  in a metabolically engineered E. coli strain  and pH (8.0) optima may be of greater interest than currently utilized enzymes . Thus, 10L6algC gene nucleotide and protein amino acid sequences can be found in GenBank with the Accession No. ON876002.The raw fosmid sequence read data can be found in the GenBank Short Read Archive (SRA) with the Accession No. SAMN29845194. The SJ, MI, MD, and PZ performed the experimental work. SJ, MD, PZ, and DD analyzed the data. All authors conceived and designed the experiments and contributed reagents, materials, and analysis tools. SJ, DD, and AD wrote the manuscript. DS, DD, and AD supervised the study. All authors contributed to the article and approved the submitted version.This work was supported by the Qingdao International Innovation Cooperation Project for Science and Technology (no. 22-3-6-ghgg-1-hz), the National Nature Science Foundation of China (no. 42006101), the Marine Biotechnology ERA/NET, NEPTUNA project (contract no. PBA/MB/15/02), the Irish Department of Agriculture, Food and the Marine (DAFM) SMI-BIO project (15/F/698), and the BlueBioCOFUND project MINERVA.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fmicb.2022.1000634/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Reminiscence therapy (RT) ameliorates psychological problems and quality of life in cancer patients. However, no study reports its effect on older papillary thyroid carcinoma (PTC) patients. This study intended to investigate the effect of the RT-care program (CP) on anxiety, depression, quality of life, and survival in older PTC patients.Eighty-six postoperative older PTC patients were enrolled and randomly assigned to RT-CP group (N = 44) and usual (U)-CP group (N = 42) as a 1:1 ratio for a 6-month intervention. Hospital Anxiety and Depression Scale (HADS) and Quality of Life Questionnaire-Core 30 (QLQ-C30) scores were assessed at baseline, month (M)1, M2, M4, and M6.p = 0.029), and HADS-depression score at M2 (p = 0.030), M4 (p = 0.029), M6 (p = 0.012) were reduced in RT-CP group versus U-CP group. Meanwhile, anxiety and depression rates from M1 to M6 were slightly decreased in RT-CP group versus U-CP group but did not reach statistical significance. Furthermore, depression severity at M6 was reduced in RT-CP group versus U-CP group (p = 0.049). Besides, QLQ-C30 global health status was increased at M2 (p = 0.023) and M6 (p = 0.033), QLQ-C30 function score was elevated at M2 (p = 0.040) and M4 (p = 0.035), while QLQ-C30 symptom score was decreased at M2 (p = 0.046) in RT-CP group versus U-CP group. Moreover, disease-free survival and overall survival were not different between two groups.HADS and QLQ-C30 scores at baseline were not different between two groups. Additionally, HADS-anxiety score at M6 (RT-CP may be a potential intervention for ameliorating anxiety, depression, and quality of life in older PTC patients. Papillary thyroid carcinoma (PTC) is the predominant form of thyroid cancer, which accounts for nearly 85% of all thyroid cancer cases . CurrentReminiscence therapy (RT) is a kind of psychotherapy aiming to ameliorate the psychological status and quality of life by recalling and sharing wonderful past memories, which is generally applied to patients with neurological diseases, such as Alzheimer’s disease (AD), dementia, acute ischemic stroke (AIS), etc. . At presAccordingly, this randomized, controlled study designed an RT-care program (RT-CP), intending to investigate the effect of RT-CP on psychological health and quality of life in older PTC patients.A total of 86 older PTC patients who received tumor resection between February 2019 and October 2021 were continuously included in this randomized, controlled study. The inclusion criteria contained: (a) pathologically confirmed as PTC; (b) age ≥ 60 years; (c) received tumor resection; (d) capable of the assessment required in the study; and (e) had willingness for participation and regular follow-up. The exclusion criteria contained: (a) complicated with other primary carcinomas or malignancies; (b) concomitant with neurodegenerative disease, or severe cognitive impairment; (c) patients who had documented diagnoses of anxiety and depression before surgery; and (d) incapable of normal communication. The study was permitted by the Ethics Committee of Harbin Medical University Cancer Hospital. Each patient signed the informed consent.Following the enrollment, eligible patients were assigned at random (1:1 ratio). The block randomization method was used to generate a random assignment table with a block size of 4. Random grouping information for each eligible patient was sealed in an opaque envelope that corresponded to the patient’s enrollment number. Based on the enrollment number, eligible patients were given the opaque envelope and then assigned to the appropriate group. As a result, 44 patients were assigned to the reminiscence therapy care program (RT-CP) and 42 patients were assigned to the usual care program (U-CP).For patients in the U-CP group, U-CP was carried out after discharge at the rehabilitation center of our hospital once every 2 weeks for 6 months. Each U-CP lasted 120 min, which mainly included health education, standardized follow-up, and answering questions. The health education included an introduction to PTC, management of postoperative complications, rehabilitation guidance, self-monitoring, precautions, medication management, diet and lifestyle management, daily exercise, and mental health.For patients in the RT-CP group, RT-CP was performed at the rehabilitation center of our hospital in small groups (7–10 patients who were recruited in the same or adjacent month for per group) once every 2 weeks for 6 months, starting from the first month after discharge. Each RT-CP lasted 120 min and consisted of two elements, which were health education and reminiscence therapy. The health education contained an introduction to PTC, management of postoperative complications, rehabilitation guidance, self-monitoring, precautions, medication management, diet and lifestyle management, daily exercise, and mental health. The reminiscence therapy contained the following 12 topics: (a) a brief self-introduction; (b) sharing an interesting childhood story; (c) sharing a memorable school story; (d) sharing scenery of hometown; (e) sharing favorite food of hometown; (f) sharing memorable travel experience; (g) sharing professional experiences; (h) sharing a personal hobby; (i) sharing a favorite sport; (j) sharing a favorite celebrity or star; (k) sharing a favorite book or music; and (l) review and summarization.At discharge (M0), 1st month (M1), 2nd month (M2), 4th month (M4), and 6th (M6) after discharge, the Hospital Anxiety and Depression Scale (HADS) and Quality of Life Questionnaire-Core 30 (QLQ-C30) scores were assessed. The HADS score was adopted to evaluate anxiety and depression, with no being 0–7 scores, mild being 8–10 scores, moderate being 11–14 scores, and severe being 15–21 scores . The QLQΧ2 test, Student t-test, or Wilcoxon rank sum test. DFS and OS were shown using Kaplan–Meier curves and analyzed by log-rank test. p < 0.05 was considered significant.The sample size was estimated based on the hypothesis that the mean value HADS for anxiety (HADS-A) score at M6 was 4.0 with a standard deviation (SD) < 3.0 in the RT-CP group and 6.0 with an SD < 3.0 in the U-CP group, respectively . The sigN = 44; receiving RT-CP for 6 months) and the U-CP group  in a 1:1 ratio. During the follow-up period, 4 patients lost to follow-up in the RT-CP group; while 3 patients lost to follow-up in the U-CP group. In both the RT-CP group and the U-CP group, anxiety, depression, and quality of life were evaluated at M0, M1, M2, M4, and M6 after discharge. Then patients were followed up without intervention until June 2022, during which DFS and OS were calculated. Finally, all available data were included in the analysis with the intention-to-treat (ITT) principle , respectively. Meanwhile, there were 14 (31.8%) males and 30 (68.2%) females in the RT-CP group, as well as 15 (35.7%) males and 27 (64.3%) females in the U-CP group (p = 0.702). Other clinical properties  stage, and postoperative radioiodine) and demographical features  were not different between the RT-CP group and U-CP group either . Furthermore, the HADS-A score (p = 0.704), anxiety rate (p = 0.950), HADS-D score (p = 0.480), depression rate (p = 0.542), QLQ-C30 global health status score (p = 0.891), QLQ-C30 functions score (p = 0.830), and QLQ-C30 symptoms score (p = 0.843) were also not different between two groups at baseline. Notably, the administration of soporifics (p = 0.541), anxiolytics (p = 0.521), and antidepressants (p = 0.511) was not different between the two groups. Specifically, 12 (27.3%) patients in the RT-CP group used soporifics and 14 (33.3%) patients in the U-CP group applied this drug. Meanwhile, 8 (18.2%) patients in the RT-CP group administrated anxiolytics, and 10 (23.8%) patients in the U-CP group applied that. In addition, 7 (15.9%) patients in the RT-CP group used antidepressants, and 9 (21.4%) patients in the U-CP group applied this drug (The mean age of older PTC patients in the RT-CP group and U-CP group was 64.3 ± 3.3 years and 65.5 ± 3.4 years (his drug .p = 0.029), while it was not different at M0 (7.0 ± 2.9 vs. 7.2 ± 3.4), M1 (6.6 ± 2.4 vs. 7.0 ± 3.1), M2 (6.0 ± 2.2 vs. 6.9 ± 2.8), and M4 (5.7 ± 2.3 vs. 6.7 ± 2.8) between the RT-CP group and the U-CP group , M4 , and M6  in the RT-CP group by comparison with the U-CP group, whereas it was not different at M0 (6.6 ± 2.8 vs. 7.0 ± 3.2) and M1 (6.1 ± 2.1 vs. 6.8 ± 2.6) between the RT-CP group and U-CP group . Depression rate was slightly reduced at M1 (22.7% vs. 33.3%), M2 (17.1% vs. 31.7%), M4 (17.5% vs. 30.8%), and M6 (12.8% vs. 30.8%) in the RT-CP group compared to the U-CP group, but did not reach statistical significance , M2 (29.3% vs. 39.0%), M4 (25.0% vs. 38.5%), and M6 (20.5% vs. 35.9%) in the RT-CP group by contrast to the U-CP group, but did not reach statistical significance . Depression severity was reduced at M6 in the RT-CP group compared with the U-CP group (p = 0.049). However, it was not different at M0, M1, M2, and M4 between the two groups  and M6  in the RT-CP group by comparison with the U-CP group; while it was not different at M0 (62.0 ± 12.8 vs. 61.5 ± 17.6), M1 (69.1 ± 14.2 vs. 64.4 ± 16.1), and M4 (75.6 ± 15.8 vs. 69.8 ± 14.7) between the RT-CP group and U-CP group  and M4  in the RT-CP group compared to the U-CP group; whereas it did not differ at M0 (63.7 ± 14.9 vs. 63.0 ± 17.4), M1 (70.1 ± 15.7 vs. 65.3 ± 18.2), and M6 (81.5 ± 10.2 vs. 77.7 ± 13.0) between the RT-CP group and U-CP group  in the RT-CP group in contrast with the U-CP group but did not differ at M0 (39.7 ± 14.9 vs. 40.4 ± 18.8), M1 (32.0 ± 12.7 vs. 36.9 ± 19.8), M4 (23.1 ± 10.9 vs. 27.1 ± 13.5), and M6 (18.8 ± 9.2 vs. 22.1 ± 12.7) between the RT-CP group and U-CP group  and HADS-D score (p = 0.024) were decreased, while the QLQ-C30 global health status score (p = 0.049) was increased in the RT-CP group compared to the U-CP group. In single/divorced/widowed older PTC patients, the HADS-A score (p = 0.003) and HADS-D score (p = 0.004) were decreased, while the QLQ-C30 global health status score (p = 0.010) was increased in the RT-CP group compared to the U-CP group. In older PTC patients with the pTNM stage of I/II, the HADS-A score was decreased in the RT-CP group compared to the U-CP group (p < 0.001; In female older PTC patients, the HADS-A score (In recent years, RT has achieved a lot of attention for mitigating anxiety and depression in cancer patients . A studyIn addition to anxiety and depression, RT also plays an essential role in improving the quality of life in cancer patients, including patients with NSCLC, gastric cancer, glioma, colorectal cancer, etc. . This stMeanwhile, this study also explored the effect of RT-CP on survival in older PTC patients. It was found that DFS and OS were not different between the RT-CP group and the U-CP group. The possible argument would be that: the determinants of prognosis mainly included age, tumor size, metastasis, thyroid gland invasion, etc.; therefore, the improvement of mental health and quality of life might have a minor or even no effect on the survival profile in older PTC patients .Further subgroup analysis suggested that the effect of RT-CP was more obvious in female, single/divorced/widowed, and pTNM stage of I/II older PTC patients. The potential reasons would be that:  female aNotably, considering that premorbid anxiety and depression would affect postoperative mental issues, it was necessary to make a clear statement to clarify this issue.  This proSeveral limitations should be noticed in this study:  the follIn summary, RT-CP may be a potential intervention for ameliorating anxiety and depression, as well as improving the quality of life, while it has no effect on prolonging survival in older PTC patients. Clinically, RT-CP as a non-pharmacological intervention can be effective in improving psychiatric problems and quality of life in older PTC patients; however, the intervention approach taken in this study is conducted in a group setting; thus, whether the individual model of this intervention is equally effective or more effective for older PTC patients requires further research to verify.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Ethics Committee of Harbin Medical University Cancer Hospital. The patients/participants provided their written informed consent to participate in this study.YL conceived and designed the experiment, analyzed the data, and revised the manuscript. LC collected and analyzed the data. XY performed data analysis and provided interpretation. XR provided technical support and analyzed and interpreted the results. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Objectives: Baise, a multiethnic inhabited area of southwestern China, is a historical malaria-endemic area with a high prevalence of G6PD deficiency. However, few studies of G6PD deficiency have been conducted in this region. Therefore, we performed a genetic analysis of G6PD deficiency in the Baise population from January 2020 to June 2021.Methods: A SNPscan assay was developed to simultaneously detect 33 common Chinese G6PD mutations. 30 G6PD-deficient samples were used for the method’s validation. Then, a total of 709 suspected G6PD-deficient samples collated from the Baise population were evaluated for G6PD status, type of mutation and effect of mutations.Results: The SNPscan test had a sensitivity of 100% [95% confidence interval (CI): 94.87%–100%] and a specificity of 100% (95% CI: 87.66%–100%) for identifying G6PD mutations. A total of fifteen mutations were identified from 76.72% (544/709) of the samples. The most common mutation was discovered to be G6PD Kaiping (24.12%), followed by G6PD Canton (17.91%), and G6PD Gaohe (11.28%). We compared the G6PD mutation spectrum among Zhuang, Han and other Southeast Asian populations, and the Zhuang population’s mutation distribution was quite similar to that in the Han population.Conclusion: This study provided a detailed G6PD mutation spectrum in Baise of southwestern China and will be valuable for the diagnosis and research of G6PD deficiency in this area. Furthermore, the SNPscan assay could be used to quickly diagnose these G6PD mutations accurately. G6PD) deficiency is one of the most common enzymatic disorders of red blood cells, with a particularly high prevalence in tropical and subtropical regions, including southern China  spans 18 kb on the X chromosome (Xq28), contains an open reading frame of 1,545 bp, and encodes 515 amino acids  tubes were used to collect blood samples, which were then brought to the lab and kept in storage at 4°C.The G6PD enzyme activity was measured by a commercial G6PD Detection kit  according to the rate method , which wAccording to the manufacturer’s recommendations, genomic DNA was extracted from all samples using a QIAamp DNA Blood Mini kit . The DNA concentration was measured using a Thermo Scientific Nanodrop™ 2000 spectrophotometer and subsequently adjusted to 50 ng/L.G6PD mutations reported in Chinese population  as follow: G6PD Gaohe (c.95A>G), G6PD Songklanagarind (c.196T>A), G6PD Asahi (c.202G>A), G6PD Chinese-4 (c.392G>T), G6PD Valladolid (c.406C>T), G6PD Liuzhou (c.442G>A), G6PD Shenzhe (c.473G>A), G6PD Mahidol (c.487G>A), G6PD Taipei (c.493A>G), G6PD Nankang (c.517T>C), G6PD Miaoli (c.519C>T/G), G6PD Mediterranean (c.563C>T), G6PD Shunde (c.592C>T), G6PD Nanning (c.703C>T), G6PD Haikou (c.835A>G/T), G6PD Viangchan (c.871G>A), G6PD Fushan (c.1004C>A/T), G6PD Chinese-5 (c.1024C>T), G6PD Beverly Hills (c.1160G>A), G6PD Santiago de Cuba (c.1339G>A), G6PD Jiangxi (c.1340G>T), G6PD Union (c.1360C>T), G6PD Canton (c.1376G>T), G6PD Yannan (c.1381G>A), G6PD Kamiube (c.1387C>T), G6PD Kaiping (c.1388G>A), G6PD Laibin (c.1414A>C), and four unnamed mutations  and two Silent mutation . The 33 G6PD mutation sites in the G6PD gene are shown in A multiplex SNPscan assays were designed to detect 33 2O and 1 μL of 30–250 ng of DNA sample. The ligation reaction was performed on an ABI 2720 thermal cycler with the following cycling program: 98°C for 2 min; 5 cycles of 95°C for 1 min, 58°C for 3 h; 94°C for 2 min, hold at 72°C. Fluorescence in multiplex After that, PCRs were run on each ligation product. Every PCR mixture was made in 20 μL containing 2× PCR Buffer, 1 μL of primer mix, 8 μL of ddH2O, and 1 μL of ligation product. The PCR program was as follows: 95°C for 2 min; 9 cycles of 94°C for 20 s, 62°C–.5°C/cycle for 40 s, and 72°C for 1.5 min; 26 cycles of 94°C for 20 s, 58°C for 40 s, and 72°C for 1.5 min; 60°C for 1 h; and hold at 4°C. Using a capillary electrophoresis system and an ABI 3730XL sequencer, PCR products were separated and identified. Raw data were analysed with GeneMapper 4.1 software , and the genotypes of each locus were determined.As shown in G6PD coding region was performed as described in our earlier research  was determined using Kozlowski’s protein isoelectric point (IP) calculator (http://isoelectric.org/). Utilizing ConSurf , we looked at the evolutionary conservation of mutant amino acid residues. The pathogenicity of these potential variants was assessed by PolyPhen-2 (Polymorphism Phenotyping v2) (http://genetics.bwh.harvard.edu/pph2/) and Sorting Intolerant from Tolerant (SIFT) web server (http://sift.jcvi.org) prediction models.The bioinformatics software used in this work was used to analyze each The data are collated in Excel. All data were statistical using SPSS 22.0. Descriptive statistics were used to estimate the accuracy.G6PD mutations reported in Chinese individuals. As shown in G6PD mutations. Additionally, the SNPscan assay could precisely distinguish double mutations, such as Canton/Viangchan, Gaohe/Kaiping and Canton/Kaiping. The created approach is dependable for identifying G6PD mutations, according to all of the evidence, detailed data are shown in A SNPscan assay was developed to detect 33 G6PD mutations were identified by the SNPcan assay in the Baise population  homozygotes and 255 heterozygotes, including 73 compound heterozygotes. The mutations of G6PD Kaiping, G6PD Canton and G6PD Gaohe were the three dominant mutations with an overall frequency of higher than 79.06%, followed by G6PD Chinese-5, G6PD Viangchan and G6PD Valladolid, with a frequency of 2.11% as a minimum, respectively. The number and frequency of various mutations are presented in Fifteen pulation . Among tG6PD Kaiping, G6PD Canton and G6PD Gaohe, accounting for 79.08% .These 709 samples were classified by ethnicity. There were 412 allele mutations and 15 variants in the Zhuang nationality, most of which were r 79.08% . Howeverr 79.08% . As showG6PD Songklanagarind, G6PD c.274C>T and G6PD Chinese-5), similar to the results predicted by PROVEAN (except G6PD Fushan). However, SIFT predicted that five missense mutations  could be tolerated, and the rest were damaging. FoldX was used to predict changes in the protein stability of G6PD  were found to increase the stability of the G6PD protein, while other missense variants were predicted to destabilize the G6PD protein. Additionally, G6PD variations. The changes in protein structure and polar bonds before and after G6PD mutation are shown in Tools from bioinformatics were used to forecast how changing an amino acid might affect how a protein function . Accordi of G6PD , and thrG6PD gene variants, the prevalence of G6PD deficiency, and the relationship between genotypes and phenotypes related to enzyme function in Baise, Guangxi Zhuang Autonomous Region. The results showed that six of the most prevalent mutations were G6PD Kaiping, G6PD Canton, G6PD Gaohe, G6PD Chinese-4, G6PD Viangchan and G6PD Chinese-5, accounting for more than 60% of G6PD-deficient alleles. This result is consistent with LinZou’s research  in the Guangxi population  (G6PD was first described by Carson in 1956 . Its cli6pd.org) . FinallyG6PD genotyping, different sorts of mutations can result in various classes of variations and exhibit various symptoms (G6PD mutations is beneficial (G6PD genotypes contributes to the study of molecular biology and genetic characterization of human populations (G6PD point mutations would be beneficial to patients, their families, the doctors who treat them, and the testing labs.Today’s G6PD deficiency diagnosis primarily uses the enzyme activity detection assay, and the main diagnosis used to avoid oxidative hemolysis cannot be other than a phenotypic test, especially in women; however, there is an added value in neficial . In addiulations . Aside fulations . The SNPulations . Numerouulations . Compareulations . Compareulations . In addi


N,N′-disalicylidene-1,2-phenylenediamine]iron(III) ) derivatives 1–4 with anionic axial ligands  and complexes 5 and 6 with neutral ligands  as well as the μ-oxo dimer 7 inhibited proliferation, reduced metabolic activity, and increased mitochondrial reactive oxygen species. Ferroptosis as part of the mode of action was identified by inhibitor experiments, together with induction of lipid peroxidation and diminished mitochondrial membrane potential. No differences in activity were observed for all compounds except 4, which was slightly less active. Electrochemical analyses revealed for all compounds a fast attachment of the solvent dimethyl sulfoxide and a release of the axial ligand A. In contrast, in dichloromethane and acetonitrile, ligand exchange did not take place, as analyzed by measurements of the standard potential for the iron(III/II) redox reaction.The [ N,N′-disalicylidene-1,2-phenylenediamine) have already been extensively studied regarding their catalytic activity [1 has been identified as the most potent compound of these series [1 caused decomposition of hydrogen peroxide similar to the Fenton reaction and supports the hypothesis that not only free iron(II) ions initiate the formation of lipid ROS and ferroptosis [1 and its μ-oxo dimer 7 in acetonitrile (ACN) and dichloromethane (DCM) [7 shifted the signal to a more anodic potential and documented strong electron donating properties [[Salophene]iron(III) complexes  and neutral  groups , and dimethyl sulfoxide (DMSO), and for biological activity in the Cisplatin-resistant ovarian carcinoma A2780cis and mammary carcinoma MDA-MB-231 cell lines. Structural modification at the salicylidene ,7,16,17 ) groups , which w1–7 is depicted in 3, Fe(NO3)3, Fe(SCN)3) yielded complexes 1–3. Dissolution of 1 together with either imidazole or 1-methylimidazole in n-butanol led to an exchange of the chlorido ligand in 1 (method b). The resulting complexes 5 and 6 precipitated from the reaction mixture iron(III)]2 + CH3O]+ (at m/z 771.15) were not detected in the spectrum of 6. The HR-MS spectrum of complex 7 (2 + H]+ form at 757.0880 (m/z) and the adduct [μ-oxo-[[salophene]iron(III)]2 + [salophene]iron(III)]+ at 1126.1217 (m/z).The lability of the ligand A was also visible by high-resolution mass spectrometry (HR-MS). The mass spectra were measured in the positive mode with an Orbitrap Elite mass spectrometer with electrospray ionization. In the spectra of all complexes , the catgand see  during tomplex 7  showed t−1. Vibrations at 1299–1316 cm−1 could be assigned to the phenolate (ν(Carom–O)), which completed the chelates at the metal center iron(III) units. Somewhat more difficult was the assignment of Oxo-bridged Fe(III) complexes show symmetric and asymmetric stretching vibrations of the Fe-O-Fe moiety with considerable variability in position and intensity. The greatest influence, however, resulted from the angle between oxygen and the iron centers ,24.1 (7 (7 in the range of 750 to 850 cm−1 (characteristic for the asymmetric Fe-O-Fe frequency) a strong and broad band at 749 cm−1, which we assigned to νas(Fe-O-Fe). Comparison of the spectra obtained with 1  and 7 . Cell-culture medium without fetal bovine serum (FBS) was used to adjust the final concentrations. The biological activity of the compounds was determined in the Cisplatin-resistant ovarian cancer cell line A2780cis and the mammary carcinoma cell line MDA-MB-231. As the complexes produced similar effects in both cell lines, only the results obtained from A2780cis cells are discussed and the data from MDA-MB-231 cells are submitted as 3H]-thymidine incorporation into the DNA is an adequate parameter to quantify antiproliferative activity. Thus, the cells were incubated for 72 h with complexes 1–7 at concentrations of 0.1 µM, 0.5 µM, and 1 µM. Cisplatin was used at 1 µM and 5 µM. As expected, the effect of Cisplatin was low and the proliferation of the A2780cis cell line was only reduced at a concentration of 5 µM to 46% . The ability of the compounds to inhibit -thymidine incorporation  for 24 h was quantified by means of a luminescence assay. Compounds activity . However1, 2, and 5 were selected and their effects on A2780cis cells were studied after incubation for 24 h by flow cytometry using Annexin V and propidium iodide (PI) staining to distinguish between viable, apoptotic, and dead cells. In the negative control, treated with the solvent DMSO only, the majority of the cells (79%) remained alive, 9% died, and 4% were apoptotic  was determined. In order to determine the kind of cell-death, A2780cis cells were simultaneously incubated for 24 h with the complexes as well as the ferroptosis inhibitor Ferrostatin-1 (Fer-1) or the necroptosis inhibitor Necrostatin-1 (Nec-1). Interestingly, Fer-1 alone (1 µM) caused 22% dead and 5% apoptotic cells; 68% of the cells were still alive . Fer-1 aThe addition of Nec-1 (20 µM) did not influence the cells compared to the solvent control . Incubat1, 2, and 5 for 24 h and thereafter stained with reduced MitoTracker®Red-H2XROS to detect mROS. Furthermore, cell-membrane proteins were visualized with wheat germ lectin and Hoechst 33342 was used for imaging the nucleus. Even if live confocal microscopy did not allow quantification, the pictures clearly indicated enhanced mROS production in complex-treated cells compared to the control cells , which acts as a scavenger of free radicals . Analysiompounds .1 (ΔΨm = 52.9%) and 2 (ΔΨm = 56.7%) showed the same effectivity, 5 caused a ΔΨm of 69.7%. All these results agree with the decreased metabolic activity  complexes were suppressed by Fer-1, it was of interest to analyze lipid peroxidation as an indication of lipid ROS. Accumulation of lipid peroxides can be visualized with BODIPY™ 581/591 C11 dye. The oxidation of the phenylbutadiene residue of the fluorophore by these species shifted the fluorescence from red to green, which is analyzed by flow cytometry. 1, 2, or 5 at concentrations of 0.1 µM and 0.5 µM for 8 h, 12 h, and 24 h, respectively, before adding the dye for 30 min. Independent of the incubation time, lipid peroxidation was low in each case at 0.1 µM (1), 11.2% (2), and 7.7% (5) of the cells were positively tested for lipid ROS after 8 h. After 24 h, lipid peroxidation decreased to control levels.A2780cis cells were treated with t 0.1 µM . At a coThe above discussed results clearly demonstrate that [salophene]iron(III) complexes generate intracellular ROS, either as mROS or in a Fenton-like reaction. Both events caused cellular stress and led to cell death. However, no differences in biological activity were observed regardless of whether the complexes had an anionic or a neutral ligand. Therefore, the influence of the axial ligand on the redox potential of the complexes was investigated. The choice of solvent was of great importance, because complete exchange of the axial ligand in the mobile phase  has already been suggested by HPLC. Thus, aprotic solvents  were selected for cyclic voltammetry investigations.DCM is a solvent without a tendency to coordinate to metals and allows the measurement of [salophene]iron(III) complexes dependent on the axially bound ligand. In contrast, DMSO is known to be a solvent with high affinity to metal complexes. It causes substitution reactions, as already demonstrated, e.g., in dichlorido platinum(II) complexes . The bin1/2) were calculated in relation to ferrocene  and are listed in 1–3 in DMSO and DCM are depicted in Electrochemical measurements were performed on 1 mM solutions of the complexes in the respective anhydrous solvent with tetrabutylammonium hexafluorophosphate  as the supporting electrolyte. Before measurement, the solution was rinsed with argon to exclude the presence of oxygen. Standard potentials  without causing an ACN/Cl exchange. The octahedral arrangement resulted in a higher electron density at the metal and increased the standard potential. The substitution of the Cl complex . 1/2 of complexes 2 and 3 bearing electron-withdrawing ligands (NO3− and SCN−) at the axial position were reduced in DCM to −502 mV and −572 mV, respectively . Acetate represents an electron-rich ligand, increasing the electron density at iron(III). EV in ACN . In DMSO5 and 6 displayed a comparable E1/2 of about −660 mV in both solvents. Dissolution in DMSO shifted E1/2 to −722 mV and 726 mV , −1376 mV (ACN), and −1403 mV (DMSO) comparable to that already described + complex was formed in each case, with E1/2 in the range of −720 to −730 mV  complexes with variable axially bound ligands. If solvents with coordinative characteristics, such as DMSO or DMF, are used, a rapid exchange takes place and a consistent solvent adduct is formed.1–7. For example, during the preparation of the stock solutions of 1 and 5, the imidazole ligand and the chloride were replaced by DMSO. Although obtained from different initial complexes, nearly identical in vitro results were received for 1–7 in various stock solutions was investigated by cyclic voltammetry and HPLC. Only in the case of 7 a timely limited proof of the initial dimer was possible, with a slow degradation to monomers. The salophene ligand, however, is chelate-bound and is not labilized by solvent molecules triazol-1-ido, 5-aminotetrazol-1-ido, 5-phenyltetrazol-1-ido, and 5-methyltetrazol-1-ido ligands were previously synthesized by Vančo et al. iron(III) complexes synthesized by Lange et al. : 12.94 , 8.94 , 7.67 , 7.49 – 7.40 , 6.98 . FT-IR (νmax (cm−1)): 3052 w, 1609 m (C=N), 1559 m, 1478 m, 1274 m, 755 s.N,N′-disalicylidene-1,2-phenylenediamine was dissolved in EtOH (0.2 mol/L) and 1 eq. of the respective iron(III) salt was added to the mixture and refluxed at 78 °C for 2 h. The mixture was cooled to rt and stored overnight. The precipitate was filtered off and washed with 10 mL of EtOH and 10 mL of MeOH. The product was dried under reduced pressure. One eq. of 1). From N,N′-disalicylidene-1,2-phenylenediamine  and iron(III) chloride anhydrous . Brown powder, yield 382 mg . FT-IR (νmax (cm−1)): 3048 w, 1603 m (C=N), 1577 m, 1379 m (C–N), 1313 m (C–O), 1232 w, 759 m, 537 m. HR-MS (MeOH): m/z calculated for C20H14ClFeN2O2: 405.0093; found: 370.0404 [M-Cl−]+. Anal. Calcd. (%) C20H14ClFeN2O2 × 0.5 EtOH; C 58.84; H 4.00; N 6.54. Found (%) C20H14ClFeN2O2 × 0.5 EtOH; C 59.00; H 4.06; N 6.61.Chlorido[salophene]iron(III) (2). From N,N′-disalicylidene-1,2-phenylenediamine  and iron(III) nitrate nonahydrate . Purification by precipitation from acetone. Black powder, yield 0.15 g, . FT-IR (νmax (cm−1)): 1705 w (NO2), 1601 m (C=N), 1577 m, 1381 m (C–N), 1316 m (C–O), 1279 s (N=O), 1003 m, 979 m (N–O) 758 m, 537 m. HR-MS (MeOH): m/z calculated for C20H14FeN3O5: 432.0283; found: 370.0416 [M-NO3−]+. Anal. Calcd. (%) for C20H14FeN3O5 × 0.5 acetone × 0.5 H2O: C 54.92; H 3.86; N 8.94. Found (%) C 54.85; H 4.19; N 9.04.Nitrato[salophene]iron(III) (3). From N,N′-disalicylidene-1,2-phenylenediamine  and iron(III) thiocyanate . Black powder, yield 0.21 g, . FT-IR (νmax (cm−1)): 2061 m, 2036 s (SCN), 1601 m (C=N), 1576 m, 1532 s, 1378 m, 1316 m (C–O), 744 s, 537 m. HR-MS (MeOH): m/z calculated for C21H14FeN3O2S: 428.0156; found: 370.0404 [M-SCN−]+. Anal. Calcd. (%) for C21H14FeN3O2S × 0.5 MeOH: C 58.12; H 3.63; N 9.46. Found (%) C 58.09; H 3.49; N 9.66.Thiocyanato[salophene]iron(III) (4): Compound 7  was dissolved in 100 mL of DCM and acetic acid  was added to the mixture and refluxed at 40 °C for 1 h. The mixture was cooled to rt and the solvent was evaporated. The solid was washed with n-butanol and EtOH and dried under reduced pressure. Dark brown powder, yield 0.21 g, . FT-IR (νmax (cm−1)): 1607 (C=N), 1578, 1531, 1525s (COO), 1481 m, 1460, 1433, 1382 (C–N), 1310 (C–O), 1189, 1147, 1031 m, 561 w, 513. HR-MS (MeOH): m/z calculated for C22H17FeN2O4: 429.0538; found: 370.0412 [M-CH3COO−]+. Anal. Calcd. (%) for C22H17FeN2O4 × 0.5 EtOH: C 61.08; H 4.46; N 6.19. Found (%): C 61.07; H 4.13; N 6.50.Acetato[salophene]iron(III) (1 was dissolved in 50 mL n-butanol and 8 eq. of the respective imidazole were added. The product precipitated from the reaction mixture. One eq. of complex 5). From 1  and imidazole . Black powder, yield 0.23 g . FT-IR (νmax (cm−1)): 3131 w (C–H), 3095 w, 1598 m (C=N), 1575 m, 1538 m, 1379 m (C–N), 1299 m (C–O), 1062 m, 1033 w, 969 w, 746,71 s, 656 w 537 m. HR-MS (MeOH): m/z calculated for C23H18FeN4O2: 438.0774 [M]+; found: 370.0410 [M-imidazole]+. Anal. Calcd. (%) for C23H18ClFeN4O2: C 58.32; H 3.83; N 11.83. Found (%):C 58.18; H 3.89; N 11.81. Imidazole[salophene]iron(III)] chloride (6). From 1  and 1-methylimidazole . Drying under reduced pressure after addition of 10 mL ACN. Black powder, yield 0.07 g . FT-IR (νmax (cm−1)): 3126 w (C–H), 3109 w, 1600 m (C=N), 1576 m, 1533 m, 1378 m (C–N), 1308 m (C–O), 1027 w, 1033 w, 969 w 748 s, 659 w, 535 m. HR-MS (MeOH): m/z calculated for C24H20FeN4O2: 452.0930 [M]+; found: 370.0418 [M-1-methylimidazole]+. Anal. Calcd. (%) for C24H20ClFeN4O2 × 0.5 ACN × 0.5 H2O: C 58.05; H 4.38; N 12.19. Found (%): C 57.86; H 4.44; N 12.06.1-Methylimidazole[salophene]iron(III) chloride  was dissolved in 200 mL EtOH and 2 g of a 29% aqueous triethylamine solution, from which 7 precipitated as a red solid. The precipitate was washed with water, methanol, and EtOH and dried under reduced pressure. Yield 0.18g . FT-IR (νmax (cm−1)): 1604 s (C=N), 1577 m, 1531 m, 1314 m (C–O), 805 m, 785 w, 749 s, 534 m. HR-MS (MeOH): m/z calculated for C40H28Fe2N4O5: 756.0759; found: 755.0880 [M+H+]+, 370.0421 [[salophene]iron(III)]+. Anal. Calcd. (%) for C40H28Fe2N4O5 × 1 EtOH: 62.94; H 4.15; N 6.99. Found (%): C 63.10; H 3.90; N 7.35.Complex Ferrocene (2 mM), the respective complex (1 mM), and the supporting electrolyte  were weighed into preheated volumetric flasks and solved in a dry solvent with the help of an ultrasound bath immediately before the measurements. About 3 mL of the solution was then transferred via syringe to a micro-cell. The solution was flushed with argon by inserting the argon line outlet for a few minutes. Thereafter, the argon line outlet was placed on top of the solution to form an argon atmosphere.x-axis (E vs. Fc) of the voltammograms were set to 0 V.Five scans (cycles) at a scan rate of 100 mV/s were performed for each measurement. Additional experiments without ferrocene were performed, to assure that ferrocene did not cover additional peaks. Calculation of the anodic and cathodic standard potentials were performed via EC-Lab. With the calculated anodic and cathodic peak potential of ferrocene, the −1), and Streptomycin (100 µg ml−1)  at 37 °C under a 5% CO2/95% air atmosphere and were passaged twice weekly. To maintain Cisplatin resistance, the A2780cis cells were incubated every second week with Cisplatin at a concentration of 1 µM. Complexes 1–3 and 5–6 were dissolved in DMSO, and complexes 4 and 7 as well as Cisplatin were dissolved in DMF. Upon dilution with RPMI 1640 cell-culture medium, the final test concentration was reached. Fer-1, Nec-1, and NAC were purchased from Sigma-Aldrich.The ovarian carcinoma cell line A2780cis was kindly provided by the Department of Gynecology, Medical University Innsbruck, Innsbruck, Austria. The mammary carcinoma cell line MDA-MB-231 was purchased from DSMZ—German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. HS-5 cells were kindly provided from the Tyrolean Cancer Research Institute. All cell lines were cultured in RPMI 1640 without phenol red  supplemented with fetal bovine serum , L-Glutamine (2 mM), Penicillin . Cells were harvested after a total incubation time of 72 h by a semiautomated device and [3H]-thymidine uptake into cells expressed as counts per minute (cpm) was measured in a scintillation counter . The proliferation in the absence of complexes (as a negative control) was set at 100% and the compound’s activity was calculated as the percentage of the control.Logarithmically growing cells were seeded in triplicates in flat-bottomed 96-well plates with a density of 1 × 10Logarithmically growing cells were seeded and treated as described above . After 72 h, the cells were examined for their metabolic activity using a modified MTT assay  according to the manufacturer’s protocol. Unspecific staining generated by the FBS-containing medium was excluded by subtracting the optical density of the cell culture medium. Metabolic activity in the absence of the compounds was set at 100%.Logarithmically growing cells were seeded and treated as described above . However, the supernatant was transferred already after 24 h into a white 96-well plate and the induction of apoptosis was recorded as caspases 3/7 activity using the Caspase-Glo 3/7 kit  according to the manufacturer’s recommendations. Values were normalized to the luminescence values of the medium control only. All values were calculated according to the caspases 3/7 activity of untreated cells, which was set at 1.4 cells in 50 µL per well. The cells were incubated at 37 °C under a 5% CO2/95% air atmosphere for 24 h with each substance at a concentration of 1 µM, without or in combination with either Fer-1 (1 µM), Nec-1 (20 µM), or NAC (1 mM). After 24 h, the cells were detached with Accutase and washed with 1× Annexin buffer. Thereafter, cells were double-stained in 50 µL of 1× Annexin buffer with 1 µL of Annexin V  conjugated to FITC dye (green fluorescence) and 1 µL of the red fluorescent dye propidium iodide , which allows discrimination between viable (Annexin V−/PI−), apoptotic (Annexin V+/PI−), and dead (Annexin V+/PI+) cells. After incubation for 15 min at 4 °C, in the dark flow, cytometric analysis was performed on a FACSCanto II .Logarithmically growing cells were seeded in flat-bottomed 96-well plates with a density of 2 × 104 cells in 50 µL per well on an 8-well Nunc™ Lab-Tek™ Chambered Coverglass dish  and incubated for 24 h together with the compounds. Immediately before analysis, 5 µL of 20 µM HEPES buffer  was added to each cavity. Subsequently, 2 µL of reduced MitoTracker®Red-H2XROS  and 2 µL of Wheat Germ Agglutinin (WGA)-Alexa Fluor™ 488 conjugate  were added and incubated for 15 min. Hoechst 33342  was added 5 min before the cells were analyzed by confocal microscopy utilizing an inverted microscope  in arrangement with a spinning disk confocal system . All the images were generated using a 40× water immersion objective .Cells were seeded in duplicates with a density of 2 × 104 cells per 50 µL and incubated for 24 h with compounds 1, 2, and 5 (0.1 µM and 0.5 µM). Thirty minutes before the end of the incubation time, 100 µL of JC-1 dye was added to each well. Cells were washed twice with 100 µL of 1× dilution buffer. Analysis was performed at 535 nm/590 nm on the Enspire Multimode Plate Reader (Perkin Elmer).The determination of the mitochondrial membrane potential was performed with the JC-1 mitochondrial membrane potential assay kit  according to the manufacturer’s instructions. The cells were seeded in triplicates at a density of 1.5 × 105) were seeded in a 12-well plate  in 4 mL of medium. After incubation at 37 °C overnight under a 5% CO2/95% air atmosphere, the compounds were added at concentrations of 0.1 µM and 0.5 µM. After 8 h, 12 h, and 24 h, the cells were detached with Accutase and centrifuged at 200 rcf for 5 min. Meanwhile, a 2.5 µM BODIPY™ 581/591 C11 staining solution  was prepared in phosphate-buffered saline (PBS)  and the cells were resuspended in 100 µL of the staining solution and incubated for 30 min at 37 °C in the dark. After centrifugation for 10 min at 200 rcf and 4 °C, the pellet was resuspended in 200 µL of PBS and immediately analyzed by flow cytometry on the FACSCanto II.Cells  complexes was stable, but the axial ligands were sensitive toward exchange reactions in strongly dissociative solvents. Measurements with traces of oxygen further showed that the complexes have a high affinity to oxygen and form a μ-oxo-bridged complex. Therefore, the nearly identical biological effects of complexes 1–3 and 5–6 might be the consequence of a transformation into the DMSO[salophene]iron(III) complex. The complexes inhibited proliferation and reduced metabolic activity in two cancer cell lines. Ferroptosis was identified to contribute to the mode of action accompanied by induction of lipid peroxidation and diminished mitochondrial membrane potential. However, identical biological activity was detected for almost all compounds. Only


Eucheuma denticulatum through boiling and using a low concentration of Ca(OH)2 is reported. Compared to the traditional method of ι-carrageenan extraction using NaOH, the reported method using Ca(OH)2 had the advantages of using 93.3% less alkali and 86.8% less water, having a 25.0% shorter total extraction time, a 17.6% higher yield, and a 43.3% higher gel strength of the product. In addition, we evaluated the gel properties and structures of ι-carrageenan products extracted by Ca(OH)2 (Ca-IC) and NaOH (Na-IC). The Fourier transform infrared spectroscopy results showed that the structures of Ca-IC and Na-IC did not change remarkably. The results of the thermogravimetric analysis and differential scanning calorimetry showed that Ca-IC had the same thermal stability as Na-IC. The results of the textural analysis showed that Ca-IC had a higher hardness and better chewiness compared to Na-IC. Rheological results indicated that Ca-IC and Na-IC exhibited shear-thinning and non-Newtonian fluid properties, whereas the viscosity of Ca-IC was less than that of Na-IC. In conclusion, this new method of ι-carrageenan extraction using Ca-IC is markedly better and yields higher quality carrageenan than the conventional method of using Na-IC.An eco-friendly method for ι-carrageenan extraction from seaweed  The gel texture properties were determined using the TA-XT plus Texture Analyzer with the following parameters: probe type, P/36R; pre/post measurement speed, 1 mm·s−1; test speed, 2 mm·s−1; deformation percentage, 50%; trigger force, 5.0 g; and compression time, 5 s.About 2% and 3%  was used to measure the rheological properties of ι-carrageenan. The type of measurement unit used for rheological testing was plate to plate.w/w] CaCl2) was prepared at concentrations of 0.2%, 0.5%, 1.0%, 1.5%, and 2.0% (w/v). In the shear mode, the temperature was fixed at 75 °C, and the shear rate was varied from 0.1 s−1 to 600 s−1.A solution of ι-carrageenan (containing 0.2% [w/v) solution of carrageenan (containing 0.2% [w/v] CaCl2) was prepared. In the frequency scan mode, the temperature was fixed at 75 °C, and the oscillation frequency increased from 0.01 Hz to 10 Hz with a strain of 1%. In the temperature mode, the scanning speed was set to 2 °C·min−1, the oscillation frequency was 1 Hz, and the strain was 1%. The temperature was increased from 20 °C to 80 °C, held for 10 min, and then decreased to 20 °C.A 1.5%  for Windows. All reported values were the average of at least three independent experiments, and the significance level was set at 0.05. ANOVA was used to evaluate the effect of single-factor optimization.2 is practical and feasible, with an improved gel strength and a higher product yield. Ca-IC showed no marked changes in structure and thermal stability compared to Na-IC. In terms of gel texture, Ca-IC had improved hardness and chewiness. Rheological results showed that both the Na-IC and Ca-IC solutions exhibited pseudoplastic fluid properties. In addition, the viscosity of the Ca-IC solution was lower, and the thermal stability of the gel was better compared to that of Na-IC. Therefore, this study concludes that the extraction of ι-carrageenan by Ca(OH)2 is industrially promising.In conclusion, the extraction of ι-carrageenan by Ca(OH)