In this study, the effects of konjac glucomannan (KGM) at different concentrations on the physiochemical and sensory properties of soy protein isolate (SPI), pea protein isolate (PPI), or peanut protein isolate (PNPI) compound gels were investigated. The results revealed that when the ratio of PNPI to KGM was 90:10, the denaturation temperature of PNPI could be significantly enhanced to 119.32°C by KGM modification. Concerning the textural and microstructural features, the amount of KGM addition had positive correlation with the hardness and chewiness of each compound gel, however, too much KGM addition will cause the unstable internal structure of the PNPI/KGM compound gels (70:30 and 60:40). Furthermore, sensory results indicated that PNPI/KGM (80:20), PPI/KGM (80:20), SPI/KGM (80:20) had great potential to be considered as prototypes for novel plant‐based products, which generated the highest acceptance scores of 5.04, 5.94, and 5.36 in each group, respectively. Physicochemical and sensory properties of konjac glucomannan (KGM) and protein isolate compound gels were studied. Adding optimal amount of KGM enhanced the thermal, textural, morphological, and sensory properties of gels. Compound gels received the highest consumer acceptance when protein to KGM was 80:20. Many foIsolated soy protein is considered as the most functional of the soy protein due to its desirable physiochemical and functional properties like providing elastic gel texture, emulsifying lipid and water, and controlling viscosity , PPI (protein content ≥80%), SPI (protein content ≥90%), and KGM were purchased from Wuhan Penglei Biotechnology Co., Ltd., and used without further treatment. All chemical reagents that used in detecting physiochemical characteristics of samples were analytical grade (Sigma‐Aldrich Co.). Subsequently, all raw materials and equipment used for conducting sensory evaluation were food grade.2.2To), peak denaturation (Td), and ending (Te) temperature were computed automatically from the thermograms using software.Thermal properties of samples were examined using a previously reported method with an adaptation , peak viscosity 1 (PV1), peak temperature 2 (PT2), peak viscosity 2 (PV2), trough viscosity (TV), trough temperature (TT), and final viscosity (FV) of the various reconstituted proteins were studied.The viscosity of each type of protein and polysaccharide conjugate was evaluated by employing Rapid Visco‐Analyzer  based on a method of Wang et al.  with sli2.4The concentration of protein and polysaccharide conjugates was 10 g/100 mL. PNPI/KGM at different ratios, 100:0, 90:10, 80:20, 70:30, 60:40, and 50:50 was included, denoted as PNPI, PNK1, PNK2, PNK3, PNK4, and PNK5, respectively. The preparation of PPI/KGM and SPI/KGM followed the same rules, and the samples were labeled as PPI, PK1, PK2, PK3, PK4, PK5, SPI, SK1, SK2, SK3, SK4, and SK5, respectively. In short, solutions were prepared by thoroughly dissolving PNPI, or PPI, or SPI powder and KGM powder in 100 mL DI water. Besides, the homogeneous solution should be prepared by constant stirring with a magnetic stirring apparatus at room temperature for 45 min. Since pH plays an important part in various factors affecting the electrostatic interactions between proteins and polysaccharides, the pH of each kind of mixed solution was modified to its optimal pH  by using 2 mol/L HCl and 10% NaOH based on previous work  following a procedure at Yuan et al.  with ada2.72.7.1The protein and polysaccharide conjugates were prepared at a constant concentration of 10 g/100 mL. PNPI/KGM at different ratios, including 90:10, 80:20, 70:30, and 60:40 denoted as PNK1, PNK2, PNK3, and PNK4, respectively. The preparation of PPI/KGM and SPI/KGM followed the same rules, and the samples were labelled as PK1, PK2, PK3, PK4, SK1, SK2, SK3, and SK4, respectively. In short, solutions were prepared by thoroughly dissolving PNPI, or PPI, or SPI powder and KGM powder in 1000 mL drinking water. Besides, a homogeneous solution was prepared by constant stirring with a blender at room temperature for 45 min. The pH of each kind of mixed solution was modified to its optimal pH  by using water with different pH. Sample were heated via using a water bath at temperature of 85°C for 40 min for PNPI/KGM  were selected based on their healthy status and frequency of consumption of plant protein products. The intensive of color, aroma, texture, and odor flavor of each type of gel sample with different ratio of protein to polysaccharide were assessed. A whole set of twelve different formulated samples  were presented simultaneously for each participant. They were required to rank the intensity of these samples for each attribute via applying an unstructured and anchored scale of 9 cm based on the “low” and “high” extremes . The statistical analysis was performed using SPSS 21 software, version 21.0 . Additionally, generalized procrustes analysis (GPA) was employed for reducing the scale effects and obtaining a consensus configuration between consumers’ sensory maps, which was performed via applying the XLSTAT statistical software, version 2021.3.1 (Addinsoft).All data reported in the analysis section on the physiochemical properties of samples were triplicate observations and were represented as a mean value ± standard deviation with one‐way analysis of variance. Duncan's Multiple Range Test was performed to measure the significant differences among the mean values (33.1To) and denaturation temperature (Td) were observed in PNK5 (99.31°C) and PNK1 (119.32°C). Among PPI group, the highest To and Td values were displayed in PPI (99.54°C) and PK3 (112.75°C). Among SPI group, the maximums of To and Td were observed in SK2 (99.63°C) and SPI (107.68°C). Besides, except from PNPI/KGM complexes, the maximum values of these thermal denaturation temperatures of PPI/KGM and SPI/KGM complexes were not significantly different from those without KGM.The thermal properties of different protein isolates complexed with different ratios of KGM are presented in Table 3.2TV), peak viscosity 2 (PV2), and final viscosity (FV) of complexed protein and KGM mixtures increased significantly (p < .05) with the addition of KGM. With the increasing percentage of KGM in the sample, PV2 in peanut and soy groups showed a similar trend, that is, increased gradually and then decreased after reaching a peak of 1882.67 cP (PNK2) and 2375.33 cP (SK3). As for pea group, PV2 gradually increased to 3414.67 cP, and then then fluctuated irregularly after the PK2. Then, the TV of PNPI group, PPI group, and SPI group ranged between 140.00 cP (PNPI) and 750.33 cP (PNK5), 229.00 cP (PPI) and 1369.33 cP (PK3), 89.33 cP (SPI), and 674.00 cP (SK3), respectively. The highest FV values were observed in PNK5 (1516.00 cP), PK4 (3017.33 cP), and SK3 (2169.67 cP), while the lowest values were all shown in the PI samples, such as 202.33 cP for peanut group, 419,67 cP for pea group, and 193.33 cP for soy group. Accordingly, TV and FV of peanut group gradually increased, and the TV and FV of pea or soy group displayed similar fluctuations. Moreover, the TT of all samples only slightly fluctuated up and down within a small range, and showed no significant difference between PNKn, PKn, or SKn (n = 1–5) samples and PI samples.According to Table 3.3The compound gels made from formulas with different ratios of protein and polysaccharides were visually observed and recorded in Figure 3.4In this experiment, each sample was investigated by two‐cycle TPA method, and the data obtained were summarized in Table 3.5The microstructure features of the compound gels containing different ratios of three types of protein isolates and KGM were presented in Figure 3.63.6.1Generalized procrustes analysis of flash profile data was shown in Figure The variable loading plot Figure  shows thThe observation score plot Figure  assessedFurthermore, the biplot Figure  represenMoreover, Figure 3.6.2The results of consumer acceptance test show in Table 44.1Td value represents that the sample has a higher degree of thermal stability and less structural damage  with KGM (polysaccharide) which demonstrated that the addition of KGM to flaxseed gum generally reduced PV, TV, and FV values  for measuring quality of protein‐based materials, but Ragaee and Abdel‐Aal  found thAll in all, an appropriate amount of KGM could be added to soy/peanut/pea protein isolates because it could significantly improve the thickening ability of its compound gels by affecting the viscosity of these three types of plant proteins. This finding was consistent with the previous results stated by Ding et al. , thus al4.3The appearance of different gels Figure  provided4.4The TPA is a simple and rapid analytical technique which is widely applied to measure how food performs during processing or handling or feels when consumer eat it , PK2 (5.94), PK3 (5.10), SK2 (5.36), SK3 (5.24), and SK4 (5.04) gels, which proved that all samples other than these six varieties were disliked by participants. This result also explained why soy protein isolate was more widely used in the food industry than the other two proteins ; formal analysis ; investigation ; validation ; writing – original draft . Wenmeng He: Formal analysis ; methodology ; software ; validation ; visualization ; writing – original draft . Baojun Xu: Conceptualization ; funding acquisition ; methodology ; project administration ; resources ; supervision ; visualization ; writing – review and editing .All authors claim that there is no conflict of interest in this manuscript.Table S1.Figure S1.Figure S2.Click here for additional data file.


Background: Type 2 diabetes mellitus (DM) and periodontitis have a bidirectional relationship that is well documented in many reviews and epidemiological studies. Periodontitis has been referred to as the sixth complication of diabetes mellitus. Various studies showed improvement in Interleukin-6 levels as well as metabolic parameters after non-surgical periodontal therapy in chronic periodontitis patients with type 2 DM. Objective: To evaluate the effect of scaling and root planing (SRP) on salivary levels of IL-6 and assessment of clinical parameters in CP patients with and without T2DM.Methods: We included 50 CP patients with well-controlled T2DM (Group I), and 50 CP patients without T2DM as controls (Group II) with evident clinical inflammation, ≥ 5mm probing depth (PD) and a relative attachment level (RAL) of ≥ 5mm. Following a brief medical and dental history plaque index (PI), gingival index (GI), gingival bleeding index (BI), PD, and RAL were recorded, and an unstimulated saliva was collected. Following SRP therapy, the clinical parameters and IL-6 levels were measured after seven days, 14 days, and 30 days. Intragroup and intergroup comparisons were carried out using a paired t-test and an independent t-test. The statistical significance was set at P < 0.05. Data were analyzed using computer software, Statistical Package for Social Sciences (SPSS) v. 22.0 .Results: Intergroup comparisons of IL-6 levels at different intervals showed a significantly higher reduction in Group II than in Group I (p=0.000). While the mean difference in the GI scores from baseline to 30 days was significantly higher in Group I patients (p=0.000), the difference in the mean PI (p=0.004), mean BI (p=0.000), mean PD (p=0.000) and mean RAL scores (p=0.000) were significantly higher in Group II patients.Conclusion: This study indicates that scaling and root planing is effective in glycemic control and also has a role to play in the level of salivary IL-6 in periodontal health and T2DM with chronic periodontitis. Elevated salivary IL-6 levels indicate periodontal inflammation which is further increased in T2DM patients. Hence, elevated IL-6 can be considered a marker of periodontal destruction. Periodontitis is one of the most common diseases of humans, generally believed to be an inevitable consequence of aging. However, we have learned over time that not all people, nor all populations, are at equal risk of developing periodontitis . AlthougThe inflammatory response to periodontal infection is characterized by an increased release of inflammatory mediators, including cytokines, chemokines, and C-reactive proteins (CRP) . InterleInitial non-surgical treatment of periodontitis with scaling and root planing (SRP) substantially reduces the clinical inflammation, probing depth (PD), bleeding on probing (BOP), and further clinical attachment level (CAL) gain. Furthermore, it is associated with a reduction in salivary IL-6 levels . SimilarThis clinico-biochemical study includes 100 chronic periodontitis patients  aged between 30-60 years chosen based on the formula n = (2 * sigma^2 / delta^2) * ^ 2 where n = required sample size per group (chronic periodontitis and glycemic controls), δ = minimum clinically significant difference in IL-6 levels between the two groups who visited the out-patient department of Periodontology, New Horizon Dental College and Research Institute, Bilaspur. We obtained approval from the Institutional Ethics Committee (NHDCRI/2018/MDS/PERI/06-ECC) and informed consent was achieved from the patients prior to the study. We included 50 CP patients with well-controlled T2DM with a glycated hemoglobin level of 6-8%, diagnosed at least three years prior to the study (Group I), and 50 CP patients without T2DM as controls (Group II). Criteria for inclusion were the presence of at least 20 natural teeth, evident clinical inflammation with ≥ 5mm probing depth (PD) in 30% of sites, and a relative attachment level (RAL) of ≥ 5mm. We excluded patients with systemic diseases including, epilepsy, hypertension, asthma, thyroid, autoimmune diseases, and cardiac diseases; patients on antibiotic therapy in the past three months and immunosuppressive therapy; history of periodontal therapy in the past six months; lactating and pregnant mothers and those with habits including smoking and chewing tobacco.All patients underwent SRP following a brief medical and dental history recording and a complete and detailed baseline clinical examination using a sterile mouth mirror, and a UNC-15 graded periodontal probe was performed. Measurement of clinical parameters including plaque index (PI), gingival index (GI), gingival bleeding index (BI), pocket depth (PD), and relative attachment level (RAL) were recorded by a single examiner at baseline (before SRP), after 7 days, 14 days, and 30 days following SRP therapy  to 7 days (3.18 ±0.15), 14 days (2.51±0.31), and 30 days (1.95±0.36) after treatment (p=0.000). Similar improvements were observed in the mean GI , mean BI , mean PD  and mean RAL (14.00±0.62mm vs. 10.01±0.59mm p=0.000) scores from baseline to 30 days after SRP. In Group II, the mean PI at baseline was 3.62±0.31, which improved to 3.13±0.30, 2.43±0.26, and 1.50±0.29 at 7, 14, and 30 days after SPR, respectively (p=0.000). Similar improvements were observed in the mean GI (p=0.000), mean BI (p=0.000), mean PD (p=0.000), and mean RAL scores (p=0.000) . Following the SRP therapy, a significant reduction in the mean PI after 7 days, 14 days, and 30 days was observed in individual groups. The results are in accordance with Aziz et al.  and TalbAlthough many indices have been proposed to assess gingival health, due to the reliability, validity, and ease of recording, the GI introduced by Loe and Silness (1963) remains the preferred choice amongst practitioners. At baseline, the mean BI was comparable between groups, while the mean GI was slightly higher in Group I than in Group II. Higher BI is correlated to increased inflammation in DM patients. Similar to studies by Acharya et al.  and AgarPD and RAL are correlated to the clinical inflammation indices in periodontitis. Increased PD and RAL correspond to advanced periodontitis and are a characteristic feature in T2DM patients. The baseline mean PD (5.28±0.77mm vs. 5.51±0.79mm) and RAL (14±0.62mm vs. 15.08±0.56mm) were comparable between groups. Literature suggests the improvement in PD and RAL post-therapy. Jain et al.  and MiraLimitations and recommendationsAlthough the positive effect of SRP on IL-6 levels in CP patients with or without T2DM is observed in our study, the study has some inherent limitations. Firstly, only well-controlled DM patients were included, which limited us in evaluating whether non-surgical treatment improves glycated hemoglobin levels. Secondly, the shorter follow-up of only 30 days might have biased the clinical indices. Awareness of the patient as being evaluated at regular short intervals might have led to conscious cleaning of teeth surfaces. Hence, further studies with longer are warranted to evaluate the effect of SRP on IL-6 and clinical parameters in patients with both controlled and uncontrolled T2DM. The result of this study indicates that scaling and root planing is effective in glycemic control and also has a role to play regarding salivary IL-6 in periodontal health and type 2 diabetes mellitus with chronic periodontitis. However, further longitudinal studies with a larger sample size are required to confirm the impact of periodontal disease with diabetes on IL-6 levels with diabetes mellitus.


In addition, NiFeLDH/A–CBp shows good catalytic performance and stability as the anode catalyst for water splitting and Zn electrowinning in alkaline electrolytes. In Zn electrowinning with NiFeLDH/A–CBp, the low cell voltage of 2.08 V at 1000 A·m−2 results in lower energy consumption of 1.78 kW h/KgZn, which is nearly half of the 3.40 kW h/KgZn of industrial electrowinning. This work demonstrates the new application of high-value-added CBp in hydrogen production from electrolytic water and zinc hydrometallurgy to realize the recycling of waste carbon resources and reduce the consumption of fossil resources.Nickel–iron-layered double hydroxide (NiFeLDH) is one of the promising catalysts for the oxygen evolution reaction (OER) in alkaline electrolytes, but its conductivity limits its large-scale application. The focus of current work is to explore low-cost, conductive substrates for large-scale production and combine them with NiFeLDH to improve its conductivity. In this work, purified and activated pyrolytic carbon black (CBp) is combined with NiFeLDH to form an NiFeLDH/A–CBp catalyst for OER. CBp not only improves the conductivity of the catalyst but also greatly reduces the size of NiFeLDH nanosheets to increase the activated surface area. In addition, ascorbic acid (AA) is introduced to enhance the coupling between NiFeLDH and A–CBp, which can be evidenced by the increase of Fe-O-Ni peak intensity in FTIR measurement. Thus, a lower overvoltage of 227 mV and larger active surface area of 43.26 mF·cm The electrochemical oxygen evolution reaction (OER) has important applications in many fields, including water electrolysis, electrochemical synthesis, Zn hydrometallurgy, etc. ,2. The ONickel–iron-layered double hydroxide (NiFeLDH) is one of the promising catalysts for OER in alkaline electrolytes, with a large specific surface area and layered structure facilitating charge exchange. However, the poor conductivity and incomplete exposure of active sites hinder the at-scale application of NiFeLDH. In order to improve the conductivity of NiFeLDH, researchers have loaded NiFeLDH nanosheets onto a conductive substrate. For example, Guofa Dong et al. synthesized LDH nanosheet arrays containing NiFe on carbon fiber cloth (CC) . In anotCBp is a product from the pyrolysis of waste tires under anaerobic conditions at 450 °C ,14,whichFurthermore, with the reduction of high-grade and high-quality sphalerite resources, the shortage of zinc resources has become acute . The recBased on the above considerations, activated CBp with high purity and specific surface area obtained through acid–base purification and KOH activation was used as a conductive substrate to prepare NiFeLDH/A–CBp through a facile in situ synthesis method. In order to improve the OER catalytic performance of NiFeLDH/A–CBp, ascorbic acid (AA) as a chelating agent was introduced to enhance the coupling between NiFeLDH and A–CBp and the related synergistic effect of NiFe atoms. The optimal catalyst with a large active surface area offered a low overpotential (227 mV) for water splitting in the alkaline electrolyte. In addition, the NiFeLDH/A–CBp catalyst largely reduced the bath voltage to reduce the energy consumption during the electrolytic process. Therefore, NiFeLDH/A–CBp, as a promising catalyst, is expected to enlarge the commercial application of Zn recovery and water electrolysis.2)2, 71334-76-4, 99%), ethyl alcohol , hydrochloric acid , and sulfuric acid  were received from Beijing Chemical Factory, China. Ammonium fluoride , iron nitrate nine hydrate (Fe (NO3)3·9H2O), nickel nitrate hexahydrate (Ni (NO3)2·6H2O), and ascorbic acid  were provided by Xilong Chemical Industry, China. Zinc oxide  and sodium hydroxide  were supplied by Beijing Chemical Factory, China. Carbon paper, foam nickel, and foam nickel molybdenum were provided by Cyber.Pyrolytic carbon black (CBp) was provided by Hunan Qiheng Environmental Protection, China. Urea (CO(NH2 atmosphere. The activated product was washed and dried to obtain activated carbon black (A–CBp).A–CBp was prepared by purification and KOH activation according to the method of a previously published paper ,28. The 3)3·9H2O, and different contents of AA were ultrasonically dispersed in 50 mL water for 30 min. Then, 3.5 mmol NH4F, 0.6 mmol Ni(NO3)2·6H2O, and 15 mmol CO(NH2)2 were added to the above solution, which was stirred for 10 min at 60 °C. The resulting mixture was heated at 120 °C for 12 h. Lastly, the samples were collected, washed, and dried. Here, the obtained catalysts with different AA contents from 0 mmol to 0.4 mmol are named AA–x mmol. Among them, the optimized OER catalyst is marked as NiFeLDH/A–CBp.NiFeLDH/A–CBp was prepared by a hydrothermal method. Firstly, mixtures with 50 mg A–CBp, 0.15 mmol Fe  layer cations. The band appearing at approximately 670 cm−1 is attributed to the stretching mode of Fe–O–Ni [−1, which is mainly derived from CBp [3 peak intensity weakens, and the peak intensities of Fe–O–Ni and M–O become stronger, indicating a stronger interface connection between NiFeLDH and CBp.The surface chemical properties of NiFeLDH/A–CBp samples with different AA contents were studied using FTIR spectroscopy, as shown in  and CBp . In addir groups . The spe Fe–O–Ni . In addifrom CBp . Among t3+ and Fe2+. For NiFeLDH/A−CBp with AA−0.1 mmol, XPS spectra displays decomposed peaks of Ni 2p at 858.4 eV, 864.2 eV, 875.9 eV, and 880.3 eV, corresponding to Ni2+ 2p3/2, Ni3+ 2p3/2, Ni2+ 2p1/2, and Ni3+ 2p1/2, respectively [3+ is difficult to further oxidize due to its high valence. The observed 4 peaks at 706.7–723.8 eV correspond to Fe2+ 2p3/2, Fe3+ 2p3/2, Fe2+ 2p1/2, and Fe3+ 2p1/2 [2+ and Fe3+, as the main valence states for NiFeLDH/ACBp, are beneficial to improving the OER performance [The XPS analysis of NiFeLDH/A−CBp with AA–0 mmol and AA–0.1 mmol were shown in ectively . Ni3+ is3+ 2p1/2 , respectformance ,55. Compformance ,56. Comp−2  vs. scan rate in dl value of AA–0.1 mmol is the largest at 43.26 mF cm−2, indicating that more active sites of the catalyst can be exposed by adjusting the amount of AA. Hence, an NiFeLDH/A–CBp catalyst with 0.1 mmol AA has more active sites and a fast charge transfer rate, indicating better electrochemical catalytic properties (W), suggesting increased conductivity to boost the electron transfer rate [For water splitting, the OER catalytic performance of NiFeLDH/A–CBp samples with different AA contents was tested in 1 M KOH. Among them, AA–0.1 mmol exhibited the lowest overpotential (227 mV) at 10 mA·cm−2 a. It has−2  [57,58,5−2 . The Cdloperties d. As seefer rate ,63,64. T−2  as an anodic catalyst  as anode is approximately 2.4 V, and the energy consumption is 2.056 kWh/Kgcatalyst c; these catalyst d, and th−2, dense, uniaxial, and nanoscale zinc powder is produced, which is easy to oxidize due to low ion mobility. With the increase of the CD to 1000 A·m−2, the deposition rate increased, promoting the growth of zinc nanoparticles. The zinc powder became flaky, and the size of the zinc powder increased to a micron level, which is closer to the requirement for commercial Zn powder. Therefore, the higher current density of 1000 A·m−2 is selected as optimal, though the electrolyzer voltage and energy consumption are a little higher than that they are at lower CD values. As the CD increases to 1500 A·m−2, the flaky zinc powder becomes thicker at a faster deposition rate, resulting in many particles gathering on the surface , indicating increased active sites, electrochemical active area, and catalytic performance. In addition, NiFeLDH/A–CBp as an anodic catalyst presented a lower electrolytic voltage (2.08 V at 100 mA·cm−2), and the energy consumption (1.782 kWh/kgZn) for Zn electrodeposition in an alkaline solution was much lower than that of 3.4 kWh/KgZn in industrial acidic electrowinning. The proposed work provides an energy-saving OER catalyst derived from CBp for water splitting and Zinc electrowinning in industrial applications.In summary, we obtained the conductive material CBp from waste tires and formed high-performance catalysts for water decomposition and zinc deposition through simple in situ synthesis. The SEM images reveal that the resulting NiFeLDH uniformly covers the surface of A–CBp. The optimized NiFeLDH/A–CBp catalyst demonstrated a higher C


Obesity, a known risk factor of Alzheimer’s disease (AD), increases the activation of microglia, leading to a proinflammatory phenotype. Our previous work shows that a high fat diet (HFD) can cause neuroinflammation and cognitive decline in mice. We hypothesized that proinflammatory activation of brain microglia in obesity exacerbates AD pathology and increases the accumulation of amyloid beta (Aβ) plaques. Presently, we tested cognitive function in 8-month-old male and female APP/PS1 mice fed a HFD, starting at 1.5 months of age. Locomotor activity, anxiety-like behavior, behavioral despair, and spatial memory were all assessed through behavioral tests. Microgliosis and Aβ deposition were measured in multiple brain regions through immunohistochemical analysis. Our results show that a HFD decreases locomotor activity, while increasing anxiety-like behavior and behavioral despair independent of genotype. A HFD led to increased memory deficits in both sexes, with HFD-fed APP/PS1 mice performing the worst out of all groups. Immunohistochemical analysis showed increased microgliosis in mice fed a HFD. This was accompanied by an increase in Aβ deposition in the HFD-fed APP/PS1 mice. Together, our results support that HFD-induced obesity exacerbates neuroinflammation and Aβ deposition in a young adult AD mouse model, leading to increased memory deficits and cognitive decline in both sexes. Obesity is linked with an earlier onset of Alzheimer’s disease (AD) and increased AD neuropathology ,2. StudiThe mPrPAPPSwe/PS1ΔE9 (APP/PS1) transgenic mouse model of AD has provided an increased understanding of progressive Aβ-dependent neurodegeneration . This moThe mPrP-APPSwe/PS1ΔE9  mouse model of Alzheimer’s Disease has a modest overexpression of the human APP transgene (~2–3 fold over non-transgenic) , which lBody composition for all groups (n = 10–14) was measured using an EchoMRI 700  before HFD exposure and at 6 months on assigned diet ,21. ChanAn open field test was used to measure locomotor activity and anxiety-like behavior for all groups (n = 12–21). Animals were removed from their home cage and placed individually into a novel arena (50.8 cm × 50.8 cm × 25.4 cm) over a 30 min period. The outer 8.9 cm of the arena was designated as the “outer” region, with the center designated as the “inner” region. Activity was collected via ANY-maze tracking software  synced with a camera. Total distance traveled and average velocity were collected for locomotor activity and time spent in the inner and outer sections were collected for anxiety-like behavior. Anxiety-like behavior was evaluated by measuring the degree of thigmotaxis, or the tendency of the mouse to remain close to walls . TherefoThe Porsolt forced swimming test was used to measure behavioral despair  for all The Y-Maze spontaneous alternation test was used to examine working spatial memory  for all Spatial memory was tested using the Barnes maze as previously described  for all Non-fasted blood glucose was measured via tail vein puncture prior to termination using a glucometer  for all groups (n = 6–12). Data were statistically analyzed using GraphPad Prism 9 software.Brain tissue for all groups (n = 4–5) were fixed in 10% neutral buffered formalin  for 48 h after collection. Tissue was then put in 20% sucrose  in phosphate-buffered saline (PBS)  solution for 48 h. Brains were then submerged in cryoprotectant , 1% polyvinylpyrrolidone 40 , in PBS) at −20 °C until the time for sectioning. Before sectioning, tissue was submerged in 20% sucrose at 4 °C for 24 h.2O2 . Sections were blocked for 15 min . Sections were incubated overnight in primary antibody  solution at 4 °C. Sections were incubated in secondary antibody  for 1 h. Artificial peroxidase  was used to block for 1 h. 3,3’-diaminobenzidine (DAB) reaction was conducted for 3 min using 4% chromogen  in substrate buffer . Sections were then mounted, coverslipped, and scanned at 40× brightfield . Densitometry of the hippocampus, cortex, and hypothalamus was performed using Fiji software and statistically analyzed using GraphPad Prism 9 software.For 40 μm sections, a sliding microtome was used. Antigen retrieval was carried out for 30 min at 78 °C . For Aβ12F4 and Aβ4G8 stains, sections were washed in 70% formic acid  for 10 min. Blocking of peroxidase was performed for 20 min using 3% HFor 5 μm sections, histology was performed by the University of Minnesota’s Comparative Pathology Shared Resource (CPSR). Tissues were embedded in paraffin, sectioned, and mounted onto slides. Tissue sections were deparaffinized, rehydrated to water, and subjected to antigen retrieval using heat and citrate buffer. Endogenous peroxidase was blocked using 3% hydrogen peroxide followed by Dako Protein Serum Block . For IBA1, the primary antibody  was incubated at room temperature for 1 h. Detection was achieved using Rabbit EnVision™+ Kit  and developed using diaminobenzidine chromogen . For Aβ4G8, the primary antibody  was incubated for 1 h at room temperature. Detection was achieved using Vector M.O.M ImmPress kit  and developed using diaminobenzidine chromogen . For Aβ12F4, the primary antibody  was incubated at room temperature for 1 h. Detection was achieved using Mouse EnVision™+ Kit  and developed using diaminobenzidine chromogen . All slides were counterstained with Mayer’s Hematoxylin. Slides were scanned at 40× brightfield . Densitometry of the hippocampus, cortex, and hypothalamus was performed using Fiji software and statistically analyzed using GraphPad Prism 9 software.1, 39 = 410.3, p < 0.0001; Males: F1, 25 = 29.22, p < 0.0001) and lean mass changes . There was no significant main effect of genotype on fat mass changes  nor lean mass changes . There was also a significant interaction between diet and genotype on fat mass changes in females but not males  and not for lean mass changes in either sex . In both females and males, post hoc tests showed that HFD groups had a larger increase in both fat mass  and genotype , with a significant interaction between the two . NC-fed AD mice had increased total distance traveled relative to NC-fed WT , but genotype did not  and there was no significant interaction between the two . As with distance traveled, NC-fed AD mice had an overall increase in average velocity compared to all other groups  and genotype F1, 73 = 4.500, p = 0.0373) significantly impacted differences in inner/outer ratio, with no significant interaction between the two . A HFD decreased inner/outer ratio in both WT (p < 0.001 vs. WT HFD) and AD mice  and total average velocity , but not inner/outer ratio . There was no significant main effect of genotype on total distance traveled , total average velocity , or inner/outer ratio . There was no significant interaction between genotype and diet on total distance traveled , total average velocity , or inner/outer ratio . Within genotypes, total distance traveled was significantly decreased in HFD-fed mice relative to NC-fed mice , but genotype did not  and there was no significant interaction between the two . In both genotypes, a HFD increased time spent immobile compared to NC-fed mice , post hoc tests did not reveal any between-group differences. There was also no significant effect of genotype , nor an interaction between genotype and diet .The Porsolt forced swimming test was used to measure behavioral despair. In females, diet impacted time spent immobile  and genotype , with no significant interaction between the two . In both genotypes, a HFD decreased spontaneous alternation percentage compared to NC-fed mice , and continued through days three  and four . In the probe test, differences in time spent in the target quadrant were attributed to diet  and genotype , with no significant interaction between the two  in both sexes. As with short-term spatial memory, post hoc tests showed that while AD mice fed NC had impaired performance relative to NC-fed WT mice  and genotype , with no significant interaction between the two . In all regions, a HFD increased IBA expression regardless of genotype  or somewhat higher  than NC-fed WT controls.Microgliosis was measured by immunohistochemical staining for IBA1 in brain sections. In females, IBA expression in AD mice was higher in all brain regions examined . In the 1, 11 = 25.94, p = 0.0003; Cortex F1, 12 = 35.93, p < 0.0001; VMH F1, 12 = 27.48, p = 0.0002) and genotype , with no significant interaction between the two . As in females, NC-fed AD mice showed higher baseline IBA than did NC-fed WT , with significant exacerbation of IBA in all three regions under HFD in both genotypes , and Aβ4G8 (Aβ17–24 deposits). The results for Aβ12F4 showed increased Aβ1–42 deposits in HFD-fed AD mice compared to NC-fed AD mice. This was seen in the hippocampus and cortex regions in both sexes (17–24 deposits in HFD-fed AD mice (1–42 deposits in particular are known to make up most of the Aβ plaques found in the AD brain [1–42 and overall Aβ deposits in multiple brain regions.To examine the effect of a HFD on neuroinflammation, immunohistochemistry was performed for IBA1. The results for IBA1 staining showed increased microgliosis in HFD and AD groups for both sexes . In femath sexes . The sam AD mice . Aβ depoAD brain . As suchOur present results provide evidence that a HFD exacerbates neuroinflammation and Aβ pathology in a young adult AD mouse model, leading to increased memory deficits and cognitive decline in both sexes. A HFD leads to an increase in microglia activation, contributing to an increase in proinflammatory cytokine signaling. This activation of microglia can lead to a decrease in Aβ uptake and clearance, leading to increased Aβ plaques. This contributes to the initiation of the cascade that eventually leads to memory impairment and cognitive decline. Early adulthood obesity has been correlated with late-life dementia independent of mid- and late-life obesity , and our


Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease; however, no specific pharmacological therapy has yet been approved for this condition. Plant-derived extracts can be an important source for the development of new drugs. The aim of this study was to investigate the effects of (E)-β-caryophyllene (BCP), a phytocannabinoid recently found to be beneficial against metabolic diseases, on HepG2 steatotic hepatocytes. Using a fluorescence-based lipid quantification assay and GC-MS analysis, we show that BCP is able to decrease lipid accumulation in steatotic conditions and to change the typical steatotic lipid profile by primarily reducing saturated fatty acids. By employing specific antagonists, we demonstrate that BCP action is mediated by multiple receptors: CB2 cannabinoid receptor, peroxisome proliferator-activated receptor α (PPARα) and γ (PPARγ). Interestingly, BCP was able to counteract the increase in CB2 and the reduction in PPARα receptor expression observed in steatotic conditions. Moreover, through immunofluorescence and confocal microscopy, we demonstrate that CB2 receptors are mainly intracellularly localized and that BCP is internalized in HepG2 cells with a maximum peak at 2 h, suggesting a direct interaction with intracellular receptors. The results obtained with BCP in normal and steatotic hepatocytes encourage future applications in the treatment of NAFLD. Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder, with an average prevalence of 25%, ranging from 13% (adult African population) to 32% (Middle East population) [In recent years, natural molecules have been demonstrated to ameliorate typical conditions of NAFLD, from steatosis to inflammation. Among them, the methyl brevifolincarboxylate, a polyphenolic compound, can reduce inflammation and oxidative stress in an hepatocarcinoma cell line  and berbCB2 receptors belong to the endocannabinoid system (ECS), a complex endogenous system involved in several physiological and pathophysiological functions. The ECS exerts regulatory control on metabolism and food intake and for this reason it represents a potential target for numerous metabolic disorders such as obesity, eating disorders, dyslipidemia and steatosis ,16. The PPAR nuclear receptors are transcriptional modulators, with each isoform having a specific location and role regarding energy homeostasis, lipid and glucose metabolism and inflammatory response . PPARα iThe interaction between BCP and PPARα and PPARγ is not as well documented as it is for CB2 receptors; however, some studies have shown that there is a direct interaction between BCP and PPARα  and probThis study aimed to investigate the anti-steatotic effects of BCP in the immortalized human hepatoma cell line HepG2, one of the cell models mostly used to induce NAFLD and to test the therapeutic effects of pharmacological and natural compounds . To mimiw/w, referred hereafter as FFAm) along with BSA .HepG2 hepatoma cells were induced to become steatotic by 24 h treatment with 0.5 mM FFA mixture, made of sodium palmitate and sodium oleate . Untreated cells and cells treated with 1% BSA only were used as controls. The CellTiter-Gloiability . TM/NucBlueTM fluorescent staining . After 24 h treatment, lipid droplets and nuclei were visualized by AdipoRedstaining A. Triglystaining B–D. p < 0.01) reduced the amount of intracellular triglycerides  decrease in triglyceride accumulation/cell, with the maximum reduction at 5 µM (corresponding to a 22% reduction of the levels of the FFAm positive control). The DNA content was used to normalize the total triglyceride values to obtain the triglyceride content per unit DNA (as a proxy for triglycerides accumulation per cell). cis-Δ9-C16:1), stearic acid (C18:0), oleic acid (cisΔ9-C18:1) and arachidonic acid (C20:4). Compared to untreated controls, steatotic HepG2 cells (p < 0.01) increase of cis-Δ9-C18:1 and C16:0 (cis-Δ9-C16:1 (p > 0.05) changes were found for C20:4 (p < 0.01) reduction (−22%) in the amount of cis-Δ9-C18:1 and C16:0 (cis-Δ9-C16:1. No changes were found for C20:4 (p > 0.05)  was consistent with that of typical NAFLD models, including myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid  B. These In order to characterize the mechanism of action of BCP in the reduction of lipid accumulation in steatotic HepG2 cells, a receptor antagonist approach was employed. In particular, we focused on cannabinoid CB2 receptors and receptors involved in lipid metabolism, i.e., PPARα and PPARγ. The specific CB2 receptor antagonist AM630, the PPARα receptor antagonist GW6471 and the PPARγ antagonist GW9662 were used at concentrations obtained from literature data ,24,25. NAt the concentration range between 500 nM and 10 µM BCP (identified as the most effective doses in previous experiments), the co-treatment of HepG2 cells with the CB2 receptor antagonist AM630 (5 µM) completely reversed BCP-driven reduction of lipid accumulation, restoring values of intracellular triglycerides comparable to that of steatotic cells A. SimilaThese results indicate that BCP is able to reduce lipid accumulation in steatotic HepG2 cells by interacting with CB2 and PPAR receptors.CNR2 gene resulted in significantly upregulated steatotic cells, when compared to untreated cells. Interestingly, co-treatment with BCP significantly reduced (p < 0.001) CB2 mRNA levels in steatotic cells, bringing CB2 expression levels closer to those of the non-pathological condition  increase in PPARα expression  for BCP ions, we found that BCP was able to cross the cell membrane and enter HepG2 cells as soon as 1 h from the beginning of the treatment, with a maximum uptake measured at 2 h. After this period, the BCP intracellular concentration decreased linearly up to 24 h after treatment . Despite recent progresses in understanding the various steps involved in the development and progression of NAFLD, no approved pharmacological treatments for this very common chronic disease are yet available . TherefoIn order to mimic NAFLD in vitro, we incubated HepG2 hepatocytes for 24 h with a mixture of oleate and palmitate, which are the most abundant monounsaturated and saturated FFA in human diet . Our steBesides emphasizing BCP ability to modify intracellular lipid composition, our data suggest that BCP effects involve the activation of CB2 as well as PPAR receptors.Pparα in mice results in enhanced liver steatosis because of the impaired oxidation of FFA [PPARα and PPARγ are ligand-activated transcription factors with pleiotropic actions in several tissues. They are critical regulators not only of fatty acid metabolism, but also of glucose metabolism, inflammation and fibrosis . The roln of FFA , underscPPARγ is involved in FFA uptake and lipogenesis and has significant anti-inflammatory properties. Early results demonstrated that its activation is steatogenic in the liver  while reAccording to our data, BCP effects are mediated both through PPARα and PPARγ. The activation of PPARα likely occurs through a direct mechanism; in support of this view, an interesting study demonstrated through a surface plasmon resonance (SPR)-BIA core system that BCP directly binds the PPARα ligand binding domain (LBD) even if it is a hydrophobic molecule and has a relatively small molecular weight compared with conventional PPAR ligands . For PPAThe binding of BCP to the CB2 receptor has been well characterized , and theWhile PPARs are well known intracellular receptors, CB2 are seven-domain transmembrane receptors, whose localization is assumed to be on the plasma membrane. To verify this assumption, we performed immunolocalization studies. Unexpectedly, we found that CB2 receptors are located mainly intracellularly in HepG2 cells, often in a perinuclear position, most likely on the ER membrane or other intracellular organelles. Recent studies have suggested possible intracellular CB2 localization in specific cell types; for instance, Castaneda and colleagues  demonstr® Luminescent Cell Viability from Promega , anti-CB2 primary antibody from Cayman chemical , anti-Rabbit IgG AlexaFluor647 secondary antibody from Jackson Immunoresearch , AM630, GW9662 and GW6741 antagonists from Cayman chemical and sodium oleate, sodium palmitate and bovine serum albumin from Sigma-Aldrich. Unless otherwise specified, all other chemicals were purchased from Sigma-Aldrich.Chemicals used were: (E)-β-caryophyllene (BCP) purchased from Sigma-Aldrich , AdipoRed™ assay reagent from Lonza , NucBlue Live ReadyProbes Reagent from Invitrogen , CellTiter-GloHepG2 human hepatoma cell line  was purchased from Sigma-Aldrich. Cells were cultured in Minimum Essential Medium Eagle (MEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/mL penicillin, 50 µg/mL streptomycin and 1% non-essential amino acids (NEAA). For every experiment, cells were grown at sub-confluence.® Luminescent Cell Viability Assay, based on the quantitation of ATP, which signals the presence of metabolically active cells. Cells were washed in phosphate-buffered saline (PBS), then CellTiter-Glo® reagent, diluted 1:1 in PBS, was added. Cells were incubated at room temperature in the dark for 10 min, then luminescence was detected and quantified with the FilterMax F5 Multi-Mode microplate reader . The values of luminescence are directly proportional to the number of viable cells. The viability of HepG2 cells was evaluated at the end of the steatosis induction experiments and treated with different concentrations of BCP, by CellTiter-Glo4 cells/well were seeded in 96-well black clear bottom plates . Prior to the experiments, cells were starved overnight with serum-free MEM, then cells were incubated for 12 h or 24 h in serum-free MEM containing 0.25 mM, 0.5 mM or 1 mM free fatty acid mixture (FFAm). The mixture was prepared by coupling sodium palmitate (Na+-hexadecanoate) and sodium oleate (Na+-(Z)-octadec-9-enoate) (1:2 ratio) with 1% w/v FFA-free BSA in serum free MEM, at 38 °C in agitation overnight, to allow FFA coupling with BSA; the mixture was then filtered and used immediately in subsequent experiments or frozen at 20 °C. Based on the preliminary results we obtained, for subsequent experiments, HepG2 cells were treated for 24 h with 0.5 mM FFAm alone or in the presence of scalar dilutions of BCP (50 nM−50 μM); control cells were grown with serum-free MEM containing 1% w/v BSA. At the end of the experiments, cells were washed in PBS, then a dye mixture containing AdipoRed and NucBlue reagents  was added. AdipoRed assay reagent quantifies intracellular triglycerides, while the DNA content was estimated by NucBlue staining. After 40 min of incubation at room temperature in the dark, fluorescence was measured with Filtermax F5 microplate reader ; for AdipoRed, quantification excitation was performed at 485 nm and emission read at 535 nm, while for NucBlue, excitation was at 360 nm and emission read at 460 nm.For steatosis induction experiments, 2 × 104 cells/well were seeded in 96-well black clear bottom plates, starved and treated as above mentioned with FFA mixture, 5 µM BCP and the following antagonists: 5 µM AM630 (CB2 receptor antagonist), 100 nM GW6471 (PPARα receptor antagonist) or 10 µM GW9662 (PPARγ receptor antagonist). For antagonists experiments, 2 × 106 cells were grown on T-25 flasks. After cell starvation, HepG2 cells were treated with FFAm alone or FFAm and 5 µM BCP for 24 h. One ml of culture medium was then taken for gas chromatography–mass spectrometry (GC-MS) analysis of free fatty acids. The intracellular fatty acid content was also analyzed; cells were washed with PBS, detached with trypsin and centrifuged at 800 g, 5 min. Lipids were extracted from cell pellets using cyclohexane  and then esterified with boron tri-fluoride (10% w/v in methanol). Fifty μg heptadecanoic acid (C17:0) was added as the internal standard. Fatty acid methyl esters (FAME) identification was performed by gas chromatography coupled with mass spectrometry (GC-MS) . FAME quantitative analyses were performed through GC coupled with a flame ionization detector (GC-FID) . The GC carrier gas was helium with a constant flux of 1 mL min−1, and separation was obtained with a non-polar capillary column ZB5-MS  . GC-FID FAME separation was performed in the same conditions, by using a similar column. Mass spectrometer parameters were: ionization energy of the ion source set to 70 eV and the acquisition mode set to 50–350 m/z. Separated molecules were identified through the comparison of mass fragmentation spectra with reference spectra of the software NIST v2.0 and libraries NIST 98, by comparison of Kovats indexes and the internal standard injection . The results are expressed as mg g−1 fresh weight (f.wt).For lipid extraction and quantification experiments, 2 × 106 cells were grown on 6-well plates and treated with 5 μM BCP. Starting from time zero and after 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 10 h and 24 h cells were washed in PBS, detached with trypsin, centrifuged and the pellets were transferred in glass vials where 1 mL of hexane was added. The identification of BCP amounts in cells at different times was performed with the Single Ion Monitoring (SIM) method by GC-MS. The following chromatographic conditions were used: column ZB5-MS ; splitless mode, oven program: 40° for 1 min, then a 5 °C min−1 ramp to 200 °C, a 10 °C min−1 ramp to 220 °C, and a 30 °C min−1 ramp to 260 °C, final temperature held for 3.6 min. Mass spectra were acquired within the 29–350 m/z interval operating the spectrometer at 70 eV and at scan speed mode. The identification of BCP was performed on the basis of both matches of the peak spectra with a library spectral database, and comparison with pure standards.For BCP time-course uptake experiments, 1 × 104 cell/cm2 were seeded on glass coverslips and incubated for at least 3 h to allow adhesion. Then, cells were fixed for 30 min in 4% paraformaldehyde dissolved in 0.1 M phosphate buffer, pH 7.3. After three washes with PBS, cells were incubated for 15 min with PBS containing 0.01% Triton-X100 and then for 45 min with PBS containing 1% BSA and 10% normal donkey serum. Triton-X100, normal donkey serum and BSA concentrations were selected after several trials to avoid autofluorescence of HepG2. Cells were then incubated overnight at 4 °C with anti-CB2 (1:100 in PBS) primary polyclonal antibody. Coverslips were washed twice with PBS and incubated for 1 h at room temperature with the secondary antibody, anti-rabbit IgG Alexa Fluor 647 . After two washes in PBS, cells were incubated for 20 min in DAPI (1:200 in PBS), washed again twice in PBS and then coverslips were mounted on standard slides with DABCO. Pictures of HepG2 cells immunolabeled for CB2 were taken with a TCS SP5 confocal microscope . Confocal image z-stacks were captured throughout the thickness of the cells and were performed with 0.7 µm optical step size using an objective 63X/1.4 NA oil immersion lens with a resolution of 8-bit, 1024/1024 pixels and 100-Hertz scan-speed . Images are shown as maximum intensity projection or single plane with reslice.For immunofluorescence experiments, 6 × 106 cells were grown on 6-well plates and treated to induce steatosis in the presence/absence of 5 µM BCP (see above). Total RNA extraction was performed using TRIZOL® Reagent (Invitrogen) following manufacturer’s instructions. Chloroform was added and, after 5 min of centrifugation at 14,000 rpm, RNA was precipitated in isopropanol for 3 h at −20 °C. Samples were then centrifuged at 14,000 rpm for 15 min, the RNA was washed with 70% ethanol and centrifuged at 14,000 rpm for 5 min. The RNA pellet was briefly air-dried and resuspended in 30 µL sterile water. Samples were quantified using NanoDrop 8000 Spectrophotometer . qReal-time PCR was performed using SensiFast SYBR No-ROX One-Step Real-Time qPCR kit  in the thermal cycler Rotor Gene Q . qRT-PCR conditions were: retrotranscription , initial denaturation , 50 cycles of denaturation , annealing , extension  and final melting (ramp from 56 °C to 99 °C). Each RNA sample was analyzed in three technical replicates containing 50 ng of total RNA. Relative quantification of mRNA abundance in each sample was performed using a standard curve, built with several dilutions of the samples. The widely used housekeeping gene β-actin was used as an internal control to normalize target gene expression. The reliability of the housekeeping gene was confirmed by its consistency of expression across treatments. qRT-PCR starting from constant amounts (50 ng) of different RNA samples, accurately quantified by NanoDrop Spectrophotometer analysis, resulted in fact in comparable levels of amplification . Specific primers were designed with PrimerBlast software on the basis of human sequences  from at least three technical replicates of 3–5 independent biological experiments (the exact number of independent experiments is indicated in the figure legends). Statistical analysis was performed using the SPSS package version 28. Statistically significant differences between treatment and control groups were assessed by a one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple-comparison post hoc test. Differences were considered statistically significant at Taken together, our results suggest that BCP is a promising molecule for the treatment of NAFLD. This conclusion is based on several key aspects of this molecule. BCP may act on multiple targets, many of which are included in NAFLD and metabolic syndrome, since it is able to reduce lipid accumulation in hepatocytes but also in adipocytes , may imp


There are no data about its effects on endometriosis, a common gynecological disease affecting 6–10% of cycling women, generating painful pelvic lesions and infertility. Current combined hormone treatment (progestins and estrogens) is safe and efficient; nevertheless, one-third of patients develop progesterone (P4) resistance and recurrence by reducing P4 receptors (PRs) levels. We aimed to compare E4 and 17β-estradiol (E2) effects using two human endometriotic cell lines  and primary cultures from endometriotic patients. We evaluated cell growth (MTS), migration (wound assay), hormone receptors levels (Western blot), and P4 response by PCR array. Compared to E2, E4 did not affect cell growth or migration but increased estrogen receptor alpha (ERα) and PRs, and reduced ERβ. Finally, the incubation with E4 improved the P4 gene response. In conclusion, E4 increased PRs levels and genetic response without inducing cell growth or migration. These results suggest that E4 might be useful for endometriosis treatment avoiding P4 resistance; however, evaluating its response in more complex models is required.Estetrol (E4), a natural estrogen produced by the human fetal liver, is actively studied for menopause and breast cancer treatment. It has low side effects and preferential estrogen receptor alpha (ER On the other hand, anti-estrogenic effects of E4 were reported in the central nervous system [Estetrol E4) is a natural estrogen produced exclusively during pregnancy by the human fetal liver . Bindingta (ERβ) . This reta (ERβ) ,4, endot is a natta (ERβ) , bone [6ta (ERβ) , and ovata (ERβ) ,9, E4 shs system  and epits system ,11. Thiss system ,12,13,14s system ,16. E4 wEndometriosis is one of the most common gynecological disorders. Around 6–10% of women of reproductive age (from the beginning of menstruation to menopause) in the western world are affected by this disease ,18. ThisA clear difference among eutopic and ectopic endometrium is their response to hormonal stimulus. Eutopic endometrium is sensitive to E2 during proliferative phase (due to increased levels of ERα); later, increments of PRs lead to P4 response during secretory phase ,35. On tConsidering its safety ,14,15, Eβ-estradiol, estetrol, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich . Ethanol was acquired from Winkler . The primary antibodies were PARP 1/2 (sc-7150) and ERα (F-10) . Progesterone receptor (6A1) , ERβ (Abx121395), and β-actin (Abx133823) .First, 17All experimental procedures were approved by the Ethical Scientific Committee at the Pontificia Universidad Católica de Chile N°170529004 and were endorsed by the Chilean National Fund of Science and Technology. All samples were collected at the Clinical Hospital of Pontificia Universidad Católica de Chile after obtaining written informed consent from the patients. By signing the informed consent, patients agreed to publish obtained results. All research was performed according to the approved guidelines and regulations.Endometrial biopsies were obtained from patients diagnosed with endometriosis who underwent laparoscopic surgery. Endometrial biopsies were taken using a sterile pipelle cannula. The protocol used was based on the previously described method . Immediag, resuspended in growth medium containing DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 0.2% gentamicin, and 0.2% amphotericin B, and seeded in T75 flasks, whereas the endometrial epithelial cells (hEEC) were retained by the strainer. The epithelial fraction was collected by backwashing the cell strainer with 15 mL of growth medium consisting of DMEM/F12 supplemented with 10% FBS, 0.2% gentamicin, and 0.2% amphotericin B, and was centrifuged at 280× g for 5 min at room temperature. Then, the pellet was resuspended in 1 mL 0.25% trypsin-EDTA and incubated for 10 min at 37 °C to dissociate any epithelial clumps. Next, 9 mL of growth medium was added to mechanically dissociate any remnant of epithelial clumps by vigorously pipetting the solution up and down. Finally, solution containing hEEC was centrifuged at 280× g for 5 min at room temperature, and pellet was resuspended and seeded in T75 flasks. Cells were kept at 37 °C in 5% CO2 and the medium was changed every 2 days.The biopsy was cut into small fragments and incubated in phenol-red free DMEM/F12 supplemented with 0.5 mg/mL collagenase type I  and supplemented with DNase I  at 37 °C with agitation for 60 minutes on a shaker. After incubation, the resultant cell mixture was shaken thoroughly and poured through a 40 µm cell strainer. The filtrate containing the endometrial stromal cells (hESC) was centrifuged for 5 min at 720× −11 to 1 × 10−7 M) [−9 to 1 × 10−5 M) [Stromal endometriotic cell line Hs832 was acquired in ATCC  and epithelial endometriotic cell line 11Z  was kind 10−7 M)  or E4 (1 10−5 M)  was perf−8 M estradiol, 1 × 10−8 M BSA-conjugated estradiol, and 1 × 10−8 M G-1 for 4–18 h. Ethanol, phosphate-buffered saline (PBS), and DMSO were used as the respective vehicle controls, 15 μL of MTS solution was added at 37 °C for 2 h and was read using photometric reading [Cells were plated into 96-well plates with fresh phenol-red free DMEM/F12 5% charcoal after E2 and E4 treatments for 24 h, 5 × 10 reading .11Z and Hs832 cells were cultured into 6 well plates until 80% confluence, and then, a vertical wound (scratch) was introduced through the cell monolayer using a fine pipette tip. Medium was replaced with fresh DMEM/F12 containing 5% charcoal treated serum with E2 or E4. Wound closure was assessed by photography at 12, 24, and 48 h and quantified with Image J software.TM Mammalian Total RNA Miniprep Kit  according to the manufacturer’s recommendations. The quantity and integrity of total RNA were determined by a Thermo Scientific Nanodrop 2000 spectrophotometer. Complementary DNA (cDNA) was generated from 2 μg RNA using random primers and M-MLV Reverse transcription . Gene expression was assessed with 50 ng of cDNA using Custom TaqMan®® Array 96-Well Fast Plates  in an Applied Biosystems 7500 thermal cycler according to the manufacturer’s recommendations. Expression data were normalized using the 2−ΔΔCt method with 18S, GAPDH, GUSB, and HRPT as endogenous reference genes.Total RNA was isolated using GenEluteTM, Roche, CHE) and protein inhibitors  plus a general metalloprotease inhibitor, BB-94 10 µM. Proteins were purified by centrifugation at 12,000× g at 4 °C for 15 min and were, subsequently, quantified. After that, 20 μg of proteins was separated by electrophoresis on a 4–20% polyacrylamide gel electrophoresis  under denaturing and reducing conditions and then, was transferred to a nitrocellulose membrane  at 350 mA for 1 h. Later, membranes were incubated with sodium citrate solution 0.01 M, pH 6.0, for 10 min at 95 °C in a water bath to expose the antigens. Next, membranes were blocked with a solution of 3% (w/v) bovine serum albumin 0.1% (v/v) Tween in Tris-buffered saline, pH 7.4, and incubated overnight with the respective primary antibodies. Finally, a second incubation took place with their respective secondary antibodies conjugated with horseradish peroxidase  diluted 1:5000 in a blocking solution for 1 h at room temperature. Peroxidase activity was detected by enhanced chemiluminescence , and the bands were quantified by densitometry using Image Studio Lite .Protein extraction was performed according to our previous work . Brieflyp < 0.05. The sample size (n) was 3 per group. We calculated it using G*Power software with the following parameters: effect size 1.5, alpha 0.05, and beta 0.9.To evaluate if data were normally distributed, the Kolmorov–Smirnov normality of Graphpad Prism 5 test  was used. Data were statistically analyzed by one-way ANOVA along with Dunnett’s post hoc test. Statistical significance was established at −11 to 10−5 M) of E2 or E4 during 24 h  of E2, and almost (except 10−8 M) all E4 concentrations  according to [Considering that after E4 incubation, PRs levels were consistently increased in endometriotic cell lines and primary cultures, we decided to evaluate if this is accompanied with an increased progesterone response by measuring expression levels of 28 genes related to progesterone response in human endometrium . Considerding to  for 6 h.The present work showed the effects of E4 in endometriotic cells, comparing epithelial and stromal type under the influence of E4 or E2. First, we noticed that E4 did not induce proliferation or migration in these cells, which represents an important finding, considering that estrogens are the leading molecule for endometriosis, an estrogen-dependent disease. Moreover, it raised the levels of PRs, which boosts the response to progesterone. The association between PRs levels and P4 response is a crucial first step, suggesting that PRs induced by E4 are functional proteins that elicit activation of PRs-related genes, required to restore P4 response in patients who developed resistance or prevent its appearance ,33. InteAn estrogenic response is a crucial event for endometriotic progression, as it is not only determined by E2 concentration but also to ERs levels ,38,39,40Regarding PRs levels, PRA is known to act as a repressor of PRB ,53, but Under our conditions, the comparison between E2 and E4 revealed interesting advantages for the natural hormone in focus of endometriosis treatment, namely: the absence of proliferation or migration, and increased ERα/ERβ ratio and PRs levels. These features highlight the major problems of endometriosis treatment, high estrogenic response and diminished sensitivity to progesterone. Nevertheless, it was necessary to determine if E4 could replicate these effects in other cellular models. For this reason, we performed primary cultures from endometriotic patients who did not receive hormonal treatment to avoid the long term effects of steroids on these cells. Primary cultures were performed successfully, but the number of cells obtained were not enough to replicate all experiments; therefore, proliferation and steroid receptors levels were chosen according to relevance and feasibility. However, this model could replicate the results for proliferation A, for boAmong this study’s strengths, we highlighted that E4, an estrogenic molecule, does not elicit proliferation or migration, which favors disease spread. Additionally, there was a consistent association between the increment in PR protein values and P4 response, which validates the methodology. However, there are limitations that we would like to establish. In vitro assay results only sometimes correlate with in vivo scenarios. The reason for this is that in vitro assays do not allow cell interaction when new metabolites secreted by these cells can elicit a plethora of responses. Additionally, in vitro assays do not consider the interaction with other cells, such as the immune system or the metabolites that the liver releases during drug metabolization. In particular, the human uterus is a highly variable tissue, from physiological to pathological. In the case of endometriosis, different studies suggested that the environment changes through disease progression, from immunological components which lead to inflammation processes from the beginning of the disease to fibrotic activity at the late phases . It is sIn conclusion, compared to E2, E4 showed exciting differences, such as not promoting proliferation or migration in all cellular models used. Moreover, the increment of PRs levels correlated with a more robust genetic response over the P4 stimulus. Both responses are significant for endometriosis treatment; first, proliferation and migration are common estrogenic effects in this tissue and are related to disease spread. Second, P4 resistance due to the reduction in PRs is the leading cause of recurrence. However, there were limitations to this study which require evaluating E4 effects in more complex models and also incorporating new variables such as inflammation, immune system, and disease origin, among others.


GATA4 leads to the loss of its expression in many cancer types. The formation of high-grade astrocytomas can be promoted by the concurrent loss of TP53 and GATA4 in normal human astrocytes. Nevertheless, the impact of GATA4 alterations with linkage to TP53 changes in gliomagenesis is poorly understood. This study aimed to evaluate GATA4 protein expression, GATA4 promoter methylation, p53 expression, TP53 promoter methylation, and mutation status in patients with primary glioblastoma and to assess the possible prognostic impact of these alterations on overall survival. Materials and Methods: Thirty-one patients with primary glioblastoma were included. GATA4 and p53 expressions were determined immunohistochemically, and GATA4 and TP53 promoter methylations were analyzed via methylation-specific PCR. TP53 mutations were investigated via Sanger sequencing. Results: The prognostic value of GATA4 depends on p53 expression. Patients without GATA4 protein expression were more frequently negative for TP53 mutations and had better prognoses than the GATA4 positive patients. In patients positive for GATA4 protein expression, p53 expression was associated with the worst outcome. However, in patients positive for p53 expression, the loss of GATA4 protein expression seemed to be associated with improved prognosis. GATA4 promoter methylation was not associated with a lack of GATA4 protein expression. Conclusions: Our data indicate that there is a possibility that GATA4 could function as a prognostic factor in glioblastoma patients, but in connection with p53 expression. A lack of GATA4 expression is not dependent on GATA4 promoter methylation. GATA4 alone has no influence on survival time in glioblastoma patients.Background: Primary glioblastoma is characterized by an extremely poor prognosis. The promoter methylation of  Glioblastoma multiforme  is the most frequent primary brain tumor in adults and is characterized by very rapid growth and an extremely short overall survival time. It is classified as either a primary—de novo—or secondary tumor that develops as a result of the progression of lower-grade malignant gliomas. The standard treatment involves surgical resection followed by radiochemotherapy (RT-CHT) and adjuvant chemotherapy (CHT) with temozolomide (TMZ). However, GBM remains an incurable disease, with a median survival time of 15 months in patients who underwent multimodality treatment . PrimaryGATA4, which encodes a transcription factor involved in the regulation of apoptosis [GATA4’s promoter leads to the loss of its expression, which has been observed in several types of cancers, such as colorectal, gastric, and lung cancers [TP53 [TP53 has been found to demonstrate abnormalities in GBM, and its regulation plays a significant role in gliomagenesis. It also has an important role in the maintenance of genomic stability, the regulation of the cell cycle, cell differentiation, death, and responding to DNA damage [TP53 mutations have been detected in 28% to 58% of glioblastoma patients [IDH-wildtype GBM, IDH-mutant GBM has a higher incidence of occurrence (27% vs. 81%) [TP53 mutations are mostly point mutations, which not only abolish the tumor suppressor function, but confer gain-of-function (GOF) properties that promote GBM malignancy [TP53 mutations can contribute to the development of GBM, additional genetic alterations, including mutations in PTEN, are necessary for the progression of this disease [Epigenetic alterations are frequent occurrences in glioblastoma pathogenesis. Epigenetics is a mechanism of gene expression regulation that is important for development and cell proliferation without any alterations in gene sequence. The identification of epigenetic modifications seems to be crucial in explaining glioma etiopathogenesis and can indicate their prognostic value. One of the epigenetic mechanisms is gene promoter methylation, which causes gene silencing. Among the genes silenced in GBM due to its promoter hypermethylation is  cancers ,6,7. AgnA damage . TP53 mupatients ,10. Compvs. 81%) . In GBM,lignancy . While T disease .GATA4 promoter methylation and alterations in TP53 and its effect on GBM development has not been defined well. The aim of this study was to evaluate GATA4 protein expression, GATA4 promoter methylation, and TP53 status  in patients with primary glioblastoma and assess the possible prognostic impact of these changes on the overall survival rate.The relationship between GATA4 expression and Thirty-one patients  with primary glioblastoma (GBM) diagnosed between 2006 and 2011 were recruited. All of their tumors were histopathologically examined and classified according to the WHO classification of tumors of the Central Nervous System (CNS) . The forGenomic DNA was isolated from formalin-fixed and paraffin-embedded tissue specimens of the patients using the QIAamp DNA FFPE Tissue Kit , according to the manufacturer’s instructions. The quality and integrity of the obtained DNA was assessed via electrophoresis on 2% agarose gel. The DNA was stored at −20 °C.GATA4 gene promoter methylation was analyzed using methylation-specific PCR (MSP). The isolated genomic DNA was modified with sodium bisulfide using the EpiTect Bisulfite Kit , according to the manufacturer’s guidelines. Converted and purified DNA was stored at −80 °C before use. As a positive and negative control, EpiTect methylated/unmethylated human control DNA (bisulfite converted)  were used. The reaction mixture (20 μL volume) contained 50–100 ng of bisulfite-treated DNA as a template, 0.5 U TaKaRaEpiTaq HS , 2.5 mM MgCl2, 0.3 mM dNTP Mixture, 1× EpiTaq PCR Buffer, and 0.3 µM of the primers. The amplification was performed in Labcycler 48 Gradient . The primer sequences were as follows: forward methylated-5′-GTATAGTTTCGTAGTTTGCGTTTAGC-3′; reverse methylated-5′-AACTCGCGACTCGAATCCCCG-3′ (product of 136 bp); forward unmethylated 5′- TTTGTATAGTTTTGTAGTTTGTGTTTAGT-3′; and reverse unmethylated 5′-CCCAACTCACAACTCAAATCCCCA-3′ (product of 142 bp).GATA4 was performed on 23 samples due to the unavailability of the material. Representative MSP results are shown in The conditions for the GATA4-methylated amplicons were: 95 °C for 15 min, 38 cycles 95 °C for 30 s, 65 °C for 1 min, 72 °C for 1 min, and 72 °C for 7 min; in addition, for the GATA4-unmethylated amplicons, the conditions were: 95 °C for 15 min, 38 cycles 95 °C for 30 s, 62 °C for 1 min, 72 °C for 1 min, and 72 °C for 7 min. Each PCR reaction included a positive control (methylated DNA), negative control (unmethylated DNA), and water. Each PCR product (6 µL) was directly loaded onto 2% agarose gel and visualized under a UV illuminator (Syngen G: BOX). Methylation-specific PCR for GATA4 protein expression was investigated immunohistochemically using a GATA4 monoclonal antibody (G-4 clone), 1:50 dilution, pH 8.0 , and processed in the EnVision system . GATA4 nuclear expression was defined as positive when the proportion of positive cells was greater than 10% A. NegatiTP53 mutation status was analyzed, as previously described [TP53 mutations were analyzed using Sanger sequencing. Four exons, 5–8, of the TP53 gene, were amplified via PCR. The following components of the reaction mixture  were used: 50 ng of DNA; 1 U Taq DNA polymerase ; 1.5 mM MgCl2; 0.25 mM dATP, dCTP, dGTP, and dTTP; and 0.5 μM primers. Amplification was performed in a GeneAmp PCR System 9700 . The primer sequences were as follows: for TP53Ex5_6, forward 5′-CACTTGTGCCCTGACTTTCA-3′ and reverse 5′-CTTAACCCCTCCTCCCAGAG-3′ (product 464 bp); for TP53Ex7, forward 5′-TCATCTTGGGCCTGTGTTATCTC-3′ and reverse 5′-GTGCAGGGTGGCAAGTGG-3′ (163 bp product); and, for TP53Ex8, forward 5′-CAAGGGTGGTTGGGAGTAGA-3′ and reverse 5′-TGCTAGGAAAGAGGCAAGGA-3′ (331 bp product). PCR was performed according to the touchdown protocol for TP53Ex5_6 and TP53Ex8: 94 °C for 5 min, 63 °C for 45 s, 72 °C for 1 min, 1 cycle 94 °C for 1 min, 61 °C for 45 s, 72 °C for 1 min, 34 cycles of 94 °C for 1 min, 60 °C for 45 s, 72 °C for 1 min, and 72 °C for 10 min; the conditions for TP53Ex7 were 94 °C for 5 min, 40 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and 72 °C for 7 min. The PCR products were purified using the MiniElute PCR Purification Kit . The sequencing method was carried out using the Big Dye Terminator v1.1 Cycle Sequencing Kit  in the GeneAmp PCR System 9700, according to classical sequencing protocols. Nucleotide sequence determination was performed using the same primers used in the initial PCR step. The resulting PCR products were purified to eliminate unincorporated primers and dNTPs using the BigDyeXTerminator Purification Kit. In the next stage, the purified products were separated using the GeneGenetic 3130xl analyzer (Applied Biosystems).escribed . TP53 muTP53 promoter methylation was analyzed using MSP. The deamination of the unmethylated cytosine of the TP53 promoter was performed with sodium bisulfite using the CpGenome DNA kit , according to the manufacturer’s protocol. DNA modified in this way was stored at −80 °C until analyses were carried out. As a positive and negative control, EpiTect methylated/unmethylated human control DNA (bisulfite converted),  were used. Methylation-specific PCR for TP53 promoter methylation was performed as previously described [TP53 promoter methylation was performed on 26 samples due to material unavailability.The presence of escribed ,17. The P53 protein expression was analyzed via immunohistochemistry. The immunohistochemical expression of p53 was studied using an anti-TP53 monoclonal antibody  processed with the EnVision (DAKO) system. The TP53 antibody labels wild-type protein, with a very short half-life and is present in small amounts in normal cells. The mutant-type p53 protein, which is the mutated form, has a longer half-life and is detected via positive staining due to a point mutation. Tumor sections were examined for p53 immunoreactivity under a microscope at 20× and 40× magnifications. The expression of p53 was considered positive when the proportion of positive cells was greater than 10% A. NegatiThe Shapiro–Wilk test was used to test the distribution of continuous variables. The distribution was found to be abnormal; therefore, these data were presented as medians followed by interquartile ranges (IQRs). Continuous variables were compared using the Mann–Whitney U test. Nominal variables were presented as numbers followed by percentages. Grubbs’ test was used to detect outliers. Spearman’s rank test was used for correlation assessment.Differences between the categorical variables were evaluated using the Chi2 test or two-tailed Fisher’s exact test. The statistical analysis was exploratory, which is why we did not perform post hoc corrections for multiple testing. For the outcome analyses, overall survival (OS) was defined as the period from diagnosis to the last follow-up, with the censoring of patients who were alive at the last follow-up. Overall survival data were presented as Kaplan–Meier survival curves and compared within subgroups using the log-rank test. Cox hazards regression analyses of OS adjusted for age were performed for each variable.p values < 0.05 were considered statistically significant.Additionally, the Multiple Correspondence Analysis (MCA) was employed to evaluate the spatial distribution of the patients according to the TP53 and GATA4 status, expression, and methylation. The analysis was performed using FactoMineR and factoextra R v.4.0.2 packages. In this study, we evaluated thirty-one samples of patients with GBM . The average age at the time of surgery was 63 ± 7.23  years old. The median survival time for glioblastoma patients was nine months (range: 0–104). The patients’ characteristics are presented in GATA4 promoter was revealed in eight patients (25.8%). GATA4 protein expression was not associated with GATA4 promoter methylation (Fisher’s exact test p = 0.4). A simultaneous lack of GATA4 protein expression and methylation of GATA4 promoter was found in three patients (9.7%). The percentage intensity of GATA4 expression was similar in GATA4-methylated tumors in comparison to GATA4-unmethylated tumors  and methylation (−) vs. GATA4 expression (+) and methylation (−) p = 0.016; GATA4 expression (−) and methylation (−) vs. GATA4 expression (+) and methylation (+) p = 0.009; GATA4 expression (−) and methylation (+) vs. GATA4 expression (+) and methylation (−) p = 0.04; GATA4 expression (−) and methylation (+) vs. GATA4 expression (+) and methylation (+) p = 0.03; TP53 mutation was detected in six (19.4%) patients, and TP53 promoter methylation was detected in three (9.7%) patients. However, no association was demonstrated between p53 expression and TP53 mutations (Fisher’s exact test p = 0.64), and there was no association between p53 expression and TP53 promoter methylation or between mutation and methylation .The expression of the GATA4 protein was detected in 15 patients (48.4%), while the methylation of TP53 promoter methylation . GATA4 promoter methylation was not associated with p53 expression, TP53 mutation, or promoter methylation . There was a strong association between GATA4 expression and TP53 mutation (p = 0.007); patients positive for GATA4 protein expression were positive for TP53 mutations in six cases in comparison to nine patients without GATA4 protein expression, whereas patients without GATA4 expression were negative for TP53 mutation in 16 cases, although none of them showed GATA4 expression without a TP53 mutation.There was no association between GATA4 protein expression and p53 expression or TP53 and GATA4. The variables’ representation demonstrated two clusters of associations: first—GATA4 expression and mutation status in TP53 along dimension 1; and second—methylation in GATA4 promoter and p53 expression along dimension 2, with a total variance of 48.1% . However, in patients positive for the GATA4 protein, p53 expression was associated with the worst outcome , but in patients positive for p53 expression, the loss of GATA4 protein expression correlated with improved prognosis , and this was related to global significance . As shown in TP53 mutations occur in the DNA-binding domain (DBD), which is encoded by exons 5 to 8, leading to the inhibition of transcription factor activity [TP53. In contrast to a previous study, we found no correlation between TP53 mutations and p53 expression [TP53 mutations. MDM2 controls the p53 expression level through a dual mechanism that involves the induction of synthesis and targeting for degradation [GATA4. This gene regulates, inter alia, organogenesis, differentiation, proliferation, and apoptosis [GATA4 silencing can be achieved through promoter hypermethylation or novel somatic mutations associated with the inter alia loss of heterozygosity (LOH) [GATA4 promoter methylation in 25.8% of patients with GBM; this is close to the value of 23.2% found in a previous study on GBM patients [GATA4-methylated tumors and GATA4-unmethylated tumors , indicating that GATA4 protein expression is not regulated by GATA4 promoter methylation in GBM. The GATA4 level may be regulated by other epigenetic mechanisms, such as microRNA, as well as miR-126, which suppresses GATA4 protein expression [GATA4 silencing in GBM can also be the result of GATA4 somatic mutations [GATA4 mutations have also been demonstrated in breast, ovarian, and lung cancers [GATA4 promoter methylation has been found to be influenced by patient age, and in one study, the gene methylation ratio varied significantly across different age groups (p = 0.027)—77% of patients were older than 60 years [p = 0.17), possibly due to the small sample size. In addition, GATA4 protein expression did not influence the overall survival rate (p = 0.19). Several publications indicate that a high level of GATA4 expression is associated with various outcomes dependent on cancer type; for example, a survival analysis indicated that a high GATA4 expression level is significantly correlated with better OS in second clinical-stage ovarian cancer. However, this assessment was for gene expression, not for protein expression [p = 0.037). However, these findings from in vitro experiments indicate that GATA4 may function as a negative regulator during carcinogenesis, suggesting that this might result from negative feedback, i.e., the elevation of the expression of GATA4 to inhibit cancerous cell differentiation and proliferation [GATA4 promoter methylation correlated with better outcomes, although, due to the small size of our study group, these results were insignificant. According to the Gene Transcription Regulation Database (GTRD), TP53 transcription is regulated by GATA4. In childhood ALL, GATA4 was revealed to regulate p53 through the transcriptional activation of MDM2, thus influencing cell cycle and apoptosis. MDM2 protein expression was increased in cells that ectopically expressed GATA4, whereas the decreased expression of p53 was found. The findings indicate that GATA4 potentially modulates apoptosis in ALL via the p53-MDM2 pathway and BCL2. [p = 0.005); however, in patients positive for p53 expression, the loss of GATA4 protein expression correlated with improved prognosis . The relationship between TP53 mutations and GATA4 expression has been described in one study regarding ovarian cancer; however, it referred to gene expression, not protein expression. However, it has been shown that this dependency was insignificant for GATA4. Nevertheless, high levels of expression of other GATA family members (GATA1-3 and GATA6) were linked to a decreased overall survival rate in patients who had TP53 mutations in ovarian cancer [GATA4 promoter methylation correlated with the worst outcomes. Hence, our data indicate a probable interaction between GATA4 and p53, which may serve as a candidate for the assessment of GBM prognosis. However, this study has several limitations: it had a small sample size, only analyzed mutations of TP53 within exons 5–8, and did not include an assessment of GATA4 mutations, which could be used to evaluate additional associations.Glioblastoma multiforme (GBM) is the most common primary human brain tumor with high mortality rates, and it exhibits multiple molecular aberrations. Despite multimodal therapy, it has a very unfavorable prognosis, hence the demand for new methods that allow more accurate diagnosis, the prediction of treatment response, and even prevention. In GBM, most activity . Thus, ipression . The lacradation . The idepoptosis ,20,21. Apatients . In contpatients . In addi cancers ,25,26. A60 years . Howevers p = 0.0—77% of pnd BCL2. . SurprisGATA4 promoter methylation, but the combination of a lack of GATA4 expression and the presence of GATA4 promoter methylation did not correlate with better outcomes in this study. In addition, GATA4 alterations had no clear prognostic value; however, the loss of GATA4 protein expression was a favorable prognostic marker of survival in patients with increased p53 expression. While the loss of GATA4 protein expression only influenced the outcome in the group with p53 expression, it nevertheless indicated improved prognosis. This indicates that GATA4 could be an important added prognostic factor in specific subgroups of patients. More research is needed to assess the interaction of GATA4 with TP53 and determine how it affects the development, prognosis, and treatment response in glioblastoma patients.In conclusion, a lack of GATA4 protein expression may not be directly associated with


Helicobacter pylori (H. pylori) there is no information regarding what transcription factors induce/upregulate the expression of CLDN6. We aimed to identify whether CREB and Yin Yang1 (YY1) regulate the expression of CLDN6 and the site where they bind to the promoter sequence. Bioinformatics analysis, H. pylori lipopolysaccharide (LPS), YY1 and CREB silencing, Western blot, luciferase assays, and chromatin immunoprecipitation experiments were performed using the stomach gastric adenocarcinoma cell line AGS. A gen reporter assay suggested that the initial 2000 bp contains the regulatory sequence associated with CLDN6 transcription; the luciferase assay demonstrated three different regions with transcriptional activity, but the −901 to −1421 bp region displayed the maximal transcriptional activity in response to LPS. Fragment 1279–1421 showed CREB and, surprisingly, YY1 occupancy. Sequential Chromatin Immunoprecipitation (ChIP) experiments confirmed that YY1 and CREB interact in the 1279–1421 region. Our results suggest that CLDN6 expression is regulated by the binding of YY1 and CREB in the 901–1421 enhancer, in which a non-described interaction of YY1 with CREB was established in the 1279–1421 region.Aberrant expression of the tight junction protein claudin 6 (CLDN6) is a hallmark of gastric cancer progression. Its expression is regulated by the cAMP response element-binding protein (CREB). In gastric cancer induced by  H. pylori LPS increases the expression of CLDN6 in AGS cells [Epithelial tight junctions are dynamic structures formed via the aggregation of several different transmembrane proteins  . ClaudinGS cells .H. pylori show up-regulation and activation of CREB that correlate with early cellular inflammation and ulceration [claudin 6 expression is regulated by CREB, amongst other transcription factors [Mongolian gerbils infected with ceration . In siliceration , and tha factors . Interes factors , a pathw factors .Diverse nuclear processes, including enhancer–promoter interactions, are regulated, among others, by CCCTC-binding factor (CTCF) . YY1 conH. pylori-induced gastric cancer, there is no information regarding what transcription factors induce/upregulate the expression of CLDN6. The aim of this paper is to identify whether CREB and YY1 transcription factors regulate the expression of CLDN6 and to identify the site where they bind to the promoter sequence.The interaction between YY1 and CREB has been established in viral infections ,35 and gCLDN6 transcription is initiated; because the number of possible binding sites was extensive, we selected sites with a minimal similitude value of 0.8 for YY1 and CREB. Nine YY1 binding sites were found, but one with a similitude value of 0.9 was in a site very close to where transcription is initiated. Concerning CREB, 17 binding sites with a similitude value between 0.80 and 0.89, and three with a similitude value greater than 0.9, were selected. Interestingly, a luciferase assay determined that none of the 0.9-value YY1 or CREB participated in the transcriptional activity after LPS stimulation (CLDN6 promoter).As YY1 interacts with CREB, a Transcription Factor Binding (TFBind) analysis was performed to determine all the possible YY1 and CREB binding sites in a promoter region close to the location where H. pylori LPS [p < 0.05). Interestingly, this increase was apparently subordinated to a significant up-regulation (p < 0.01) of YY1 and CREB transcription factors that initiated 4 h after LPS exposure and reached its maximum value after 12 and 24 h   A. Transf−901 bp) B. The ap−901 bp) C. The diCLDN6 transcription regulation, silencing experiments using specific siRNAs were done. p < 0.0001).To determine the role of YY1 and CREB in As the luciferase analysis had already shown, the 901–1421 region possessed the elements that control CLDN6 expression in LPS-treated cells. The chromatin immunoprecipitation assays showed YY1 and CREB occupancy sites in the 901–1018 bp fragment; the 1018–1149 fragment only had YY1 occupancy, and the 1149–1279 fragment did not show occupancy signals for either transcription factor; these sites corresponded to those predicted via the bioinformatic analysis. The 1279–1421 fragment showed CREB occupancy as predicted, but surprisingly, YY1 occupancy was also demonstrated despite not being predicted via the bioinformatic analysis .Because of the surprising presence of a YY1 occupancy site in 1279–1421, we looked for possible YY1 binding sites with a minimal similitude value of 0.5 onwards in this region; the analysis did not show a putative binding site. Because ample bibliographic information validates the spatial interaction of both proteins, we performed sequential ChIP experiments in all three regions to determine whether the presence of YY1 in the 1279–1421 region is secondary to its interaction with CREB. The results confirmed, as expected, that YY1 and CREB interact in the 1279–1421 region, thus confirming our hypothesis .claudin 6 promoter [Claudins, a large family of transmembrane structural proteins, are fundamental to tight junction function and regulation linked to associated regulatory and scaffolding proteins . They expromoter . CLDN6 epromoter ,43,44. Cpromoter ,19, but CLDN6. The bioinformatics analysis results showed that CREB and YY1 have 17 and 9, respectively, probable binding sites 2000 bp upstream of the site where transcription of CLDN6 is initiated. Our results confirmed that H. pylori LPS induced, in the context of a chronic inflammatory process, the expression of CLDN6 in the AGS cell line [A previous analysis of claudin expression in the AGS cell line suggested that the transcription factors CREB and YY1 could be regulators of ell line . Still, YY1 is a zinc finger protein that can activate or repress transcription, depending on its interactions with other transcription factors ,46,47, sH. pylori LPS. lL-1b and TNFα regulate gene transcription via the C/EBP1, C/EBP2, and YY1 elements in epithelial cells [H. pylori capable of inducing gastric carcinogenesis; cytotoxin-associated gene A (CagA) protein, another major and substantial component of H. pylori, is known to provoke genomic hypermutations, DNA double strand breaks, subverted DNA damage responses that include the down-regulation of DNA repair genes, and transcriptomic and proteomic alterations that increase the chance of generating a “hit-and-run mechanism” for gastric carcinogenesis [Bacterial LPS regulates gene transcription by binding YY1 complexes to the CCAAT enhancer binding protein 1 (C/EBP1) or long non-coding RNA (lncRNA) via inflammatory cytokines ,52. Our al cells . YY1 funal cells . LPS is ogenesis ,56,57.CLDN6. The ChIP assays confirmed the results and revealed a YY1 binding site in the 1279–1421 bp region not predicted via the bioinformatics analysis. The distance between these CREB and YY1 sites was identified, suggesting that the 1279–1421 region is an enhancer [CLDN6 transcription, but the precise mechanism remains to be solved. YY1 has been recognized as a structural regulator of enhancer–promoter loops [To identify the specific regulatory regions of the 2000 bp promoter regions where CREB and YY1 binding sites were found, the complete promoter region and two fragments were cloned in the pMetLuc2 vector and evaluated via a luciferase activity assay. The results suggested that two CREB sites and two YY1 sites in the DNA fragment corresponding to the 901 bp–1421 bp region are significantly important for regulating the enhancer . Gene exenhancer . Enhanceenhancer . YY1 is enhancer . Our reser loops . YY1 ander loops .H. pylori LPS is involved in the transcriptional initiation and activation of CLDN6, a perfectly defined tight junction protein associated with enhanced gastric cancer progression and invasiveness [The mechanism through which YY1 functions both as a transcriptional activator and repressor depends in its acetylation/deacetylation . Still, siveness .Our results suggest that CLDN6 expression, a protein associated with gastric epithelial cancer progression, is regulated by the binding of YY1 and CREB in the 901–1421 enhancer in which a non-described interaction of YY1 with CREB was established in the 1279–1421 region.Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), L-glutamine, sodium pyruvate, insulin, Dulbecco’s phosphate-buffered saline (PBS), streptomycin–penicillin, bovine albumin, and total antibody compensation beads were from Invitrogen . Monoclonal anti-claudin-6 (sc-393671), anti-YY1 (sc-7341), anti-GAPDH (sc-47724), anti-actin (sc-32251), and goat anti-mouse IgG were from Santa Cruz Biotechnology . Monoclonal anti-CREB (MA1-083) and Lipofectamine 2000 (11668027) were from ThermoFisher Scientific . The Molecular Biology Kit, EZ-10 Spin Column Plasmid DNA Miniprep Kit was from Southern Labware . The ready-to-glow secreted luciferase reporter assay Cat # 631727) and pMetLuc2 reporter vector (Cat # 631729) were from Takara Bio USA . Primers were designed using TFBIND [1727 and 6) were cultured in sterile P-100 Petri dishes with DMEM supplemented with 5% FBS, 0.1 U/mL of insulin, a 1% streptomycin–penicillin solution, 2 mmol/L of L-glutamine, and a 2 mmol sodium pyruvate solution at 37 °C in a humid environment containing 5% CO2, until reaching >90% confluence. Luciferase experiments were performed with 8 × 105 cells/well seeded in 6 well Tissue Culture Plates  for 48 h until they reached 90% confluence, which represents 1 × 106 cells/well. The LPS treatment of AGS cells was performed in p-100 culture dishes once they reached 80–90% confluence. All the experiments were immediately performed with these confluent AGS cell cultures in their third passage.Human gastric adenocarcinoma (AGS)  cells . We used a 0.8 cut-off instead of the 0.5 value for both transcription factors.The search for sequences with potential transcriptional relevance in the present study was performed using TFBIND  were used to transfect AGS cells. 3 × 10Helicobacter pylori (strain J99) LPS were kindly donated by Dr. Victor R. Coria, Instituto Nacional de Pediatría, México. AGS cells were treated with 10 ng/mL of LPS H. pylori for 4 h, 8 h, 12 h, 24 h, and 48 h.g (4 °C). The supernatants were recovered, and a Bio-Rad Protein Assay determined the total protein concentration .Control, LPS-treated, and transcription factors silenced AGS cells were washed twice with PBS and scraped with 1 mL of an ice-cold lysis buffer (150 mM NaCl/50 mM Tris/1 mM EGTA/1 mM EDTA/1% IGEPAL/0.1% Sodium deoxycholate/0.1% SDS + protease and phosphatases inhibitors). Cell suspensions were sonicated for 1 min at 25% amplitude, followed by centrifugation for 30 min at 17,000× For Western blot analysis, 20 µg of protein was resolved on 13% SDS/PAGE and transferred to nitrocellulose membranes in a Bio-Rad semi-dry blotting system for 1 h at 120 mA. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) for 1 h, washed twice with TBS, and incubated with the relevant primary antibody diluted in Tween 20/TBS (TTBS) overnight at 4 °C (anti claudin-6 (1:200), anti-YY1 (1:400), anti-CREB (1:500), anti-GAPDH (1:5000), and anti α-actin (1:500)). Membranes were washed with TTBS and incubated with horseradish peroxidase-labeled secondary goat anti-mouse IgG (1:4000) diluted in TTBS for 1 h at room temperature. Afterward, membranes were washed thrice with TTBS and once with TBS before antibody binding was detected via chemiluminescence using the Supersignal West Dura Extended Duration Substrate  as a substrate. Equal protein loading was confirmed in all the experiments by determining β-actin as a loading control. All experiments were done in triplicate. The quantitative analysis of the Western blot bands was performed using ImageJ v 1.53 software, which calculates the intensity of the gel band, measuring the amount of pixels/sq.in. The values were determined for each band. The results are expressed as dots per point (DPP) and intensity.claudin 6 promoter were amplified from AGS genomic DNA using the following primers: −2000 forward: 5′CCAGCCGGTGATCTAGTCC3′; −1421 forward: 5′GCCACTACAGCTTTGTTAAGGG3′; and 901 forward: 5′CGGGCACCTGTAGTAGTCC3′. We used the same reverse primer: 5′AATTCCTAGGCCGAGTGTCG3′. The amplified DNA was cloned in pMetLuc2 plasmid using DNA ligase T4. The result of this ligation was used to transform competent E. coli DH5α to get transformed colonies. Plasmid DNA was extracted using the Southern Labware EZ-10 Molecular Biology Kit, according to the manufacturer´s instructions. The purified DNA was used to transfect AGS cells using lipofectamine 2000; these transfected AGS cells were treated with H. pylori LPS 10 ng/mL for 4, 8, 12, 24, and 48 h. Luciferase activity was measured using the Ready-to-Glow secreted luciferase reporter assay  in a Perkin Elmer Wallac 1420 Victor Spectrophotometer. The values obtained from these experiments were normalized according to Schagat T. of Promega Corporation .The −2000, −1421, and −901 regions of the H. pylori LPS 10 ng/mL for 24 h before being fixed with 3.7% formaldehyde, lysed, and sonicated, as previously mentioned. Immunoprecipitation was then performed with CREB and YY1 antibodies, and purified DNA fragments were analyzed via qPCR, using Ruby Hot Start Master (2×) according to the manufacturer’s instructions. The following primers were used: 901–1018 forward: 5′GCACTACAGCTTTGTTAAGGG3′; reverse: 5′CACTACCACGCCCGGCTAAC3′; 1018–1149 forward: GTTAGCCGGGCGTGGTAGTGGGC; reverse: CGCCCAGTCTGGAGTGCAATGG; 1149–1279 forward: CCATTGCACTCCAGACTGGGCG; reverse: TTTCCTGACCTCGTGATCTGCCC; and 1279–1421 forward: GGGCAGATCACGAGGTCAGGAAA; reverse: GGGACTACTACAGGTGCCCG. The results of these assays were analyzed in a 3.5% agarose gel, and the quantitative analysis of the bands’ images was performed using ImageJ v 1.53 software, which calculates the intensity of the gel band, measuring the amount of pixels/sq.in. The values were determined for each band. The results are expressed as dots per point (DPP) and intensity.A ChIP assay was performed using Millipore EZ-ChIP kit  according to the manufacturer’s instructions. In brief, AGS cells were treated with p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) or p < 0.0001 (****) versus the control condition, and “n” represents the number of independent experiments.Statistical analyses were performed using the GraphPad Prism software, version 4 . All values are expressed as means ± standard deviations (SDs). Statistical significance in a one-way analysis of variance (ANOVA), followed by a post hoc Dunett’s test and selected pairs comparison test, was set to