Zea mays. The resulting thiourea-capped apatite nanostructure (TU–ANP) was characterized using complementary analytical techniques, such as EDX, SEM, XRD and IR spectroscopy. The pre-sowing of soaked seeds of Zea mays in 1.00 µg/mL, 5.00 µg/mL and 10 µg/mL of TU–ANPs yielded growth under 0 mM, 60 mM and 100 mM osmotic stress of NaCl. The results show that Ca and P salt acted as precursors for the synthesis of ANPs at an alkaline pH of 10–11. Thiourea as a source of nitrogen stabilized the ANPs’ suspension medium, leading to the synthesis of TU–ANPs. XRD diffraction analysis validated the crystalline nature of TU–ANPs with lattice dimensions of 29 nm, calculated from FWHM using the Sherrer equation. SEM revealed spherical morphology with polydispersion in size distribution. EDS confirmed the presence of Ca and P at a characteristic KeV, whereas IR spectroscopy showed certain stretches of binding functional groups associated with TU–ANPs. Seed priming with TU–ANPs standardized germination indices  which were significantly declined by NaCl-based osmotic stress. Maximum values for biochemical parameters, such as sugar (39.8 mg/g at 10 µg/mL), protein (139.8 mg/g at 10 µg/mL) and proline (74.1 mg/g at 10 µg/mL) were recorded at different applied doses of TU–ANP. Antioxidant biosystems in the form of EC 1.11.1.6 catalase (11.34 IU/g FW at 10 µg/mL), EC 1.11.1.11 APX (0.95 IU/G FW at 10 µg/mL), EC 1.15.1.1 SOD (1.42 IU/g FW at 5 µg/mL), EC 1.11.1.7 POD (0.43 IU/g FW at 5 µg/mL) were significantly restored under osmotic stress. Moreover, photosynthetic pigments, such as chlorophyll A (2.33 mg/g at 5 µg/mL), chlorophyll B (1.99 mg/g at 5 µg/mL) and carotenoids (2.52 mg/g at 10 µg/mL), were significantly amplified under osmotic stress via the application of TU–ANPs. Hence, the application of TU–ANPs restores the growth performance of plants subjected to induced osmotic stress.Salinity is one of the most prevalent abiotic stresses which not only limits plant growth and yield, but also limits the quality of food products. This study was conducted on the surface functionalization of phosphorus-rich mineral apatite nanoparticles (ANPs), with thiourea as a source of nitrogen (TU–ANPs) and through a co-precipitation technique for inducing osmotic stress tolerance in  Zea mays is cultivated on an area of about 896 thousand hectares with a yearly mass production of 4.9 million tons of grains [Maize is grown worldwide and is the third major crop after rice and wheat, named as “king of grain crops” . The USAf grains . It is wf grains . In Pakif grains .Recent trends in agriculture have validated the use of plant growth regulators, including thiourea, to surge the growth and yield of crop plants. Thiourea (TU) not only provides a nitrogen source, but also augments tolerance against various abiotic stresses due to its high degree of solubility and rapid absorption into biosystems . EarlierZea mays via germination and seedling foliar application.Nanoparticles have unique physicochemical properties, which are used for different purposes in life sciences, chemical engineering, medicines and agriculture . Hence, −1 correspond to N-H stretching; and O-H, C-N and C=S are associated with thiourea and vibrational bending at 1032 and 520 wavenumbers cm−1, representing PO43− characteristics in the apatite-validating capping of thiourea on the surface of ANPs  effect of TU–ANPs on GP, MGT, GI and T50. Seed grown under osmotic stress of 100 mM NaCl delayed the onset of germination to 6.12 days, compared to the non-saline condition (3.18). Seeds primed with 10 µg/mL of TU–ANPs induced early germination with a T50 value of 2.98 days under the non-saline condition whereby, under 100 mM salt stress, 3.67 days were taken to reach 50% germination  dose-dependent decline in leaf area, leaf fresh and dry biomass, due to NaCl-induced osmotic stress. Foliar application of TU–ANPs at a dose of 5.0 µg/mL exhibited 1.5 g/leaf FW, 0.22 g/leaf DW and 40.87 cm2 leaf area in plants grown under the highest experimental dose of osmotic stress. Moreover, maximum shoot length (8.7 cm) was also recorded in plants with a foliar application of 5 µg/mL under 60 mM of salinity, followed by hydro-primed plants (8.5 cm) of the non-saline condition . A minimal chlorophyll b content of 39.15 mg/g was recorded in plants subjected to 100 mM NaCl-based osmotic stress, whereby a maximum chlorophyll b content of 53.51 mg/g was recorded in plants treated with 10 µg/mL of TU–ANPs via the foliar route grown under the non-saline condition. This was followed by 50.20 mg/g in plants treated with 5 µg/mL of TU–ANPs. Similarly, chlorophyll a content in plants was also subjugated to decline due to osmotic stress in a dose-dependent manner . An almoOsmotic stress inducing oxidative stress with the production of oxidative free radicals led to a decline in plant growth. TU–ANPs significantly coped with free radicals by triggering the antioxidative biosystems of plants, hence optimizing the activity of antioxidant enzymes. The activity of SOD was significantly enhanced from 30.28 IU/G FW in non-saline plants to 49.48 IU/g FW in plants subjected to 100 mM NaCl-induced osmotic stress. Foliar application of TU–ANPs at 10 µg/mL significantly reinstated the activity of SOD to 37.7 IU/g FW in plants under the highest experimental osmotic stress . A similGP but also delays the time to germination (MGT and T50). This is due to its negative effect on mitotic cell division and cell elongation, altering enzyme activity particularly associated with protein synthesis, as well as growth hormones [NaCl-based osmotic stress not only causes a reduction in hormones . Figure hormones ,20.2O2), nascent oxygen, hydroxyl free radicals (HO*) and superoxide anion (O2−) which are not only cytotoxic, but also cause damages to vital biomolecules  through oxidation, hence effecting the overall growth performance of plants [Plant agronomic traits were significantly declined by soil salinity due to nutritional imbalances in plants caused by salt ions through competitive absorption distribution and the transport of micro- and macro-minerals . Salinitf plants .2 assimilation, and consequently reducing the efficiency of thylakoids and mitochondrial electron transport [Vicia faba [Similarly, high soil–salt content inhibits the activity of RUBisCO and ATPase, causing the reduction in COransport . Plant aransport . Mitigatcia faba . Moreovecia faba .+/K+ channels, thus coping with the detrimental effects of salinity [Additionally, nano-fertilizer encouraged enzyme activities in plants; as a result, plant cells preserved from injury of reactive oxygen species (ROS) . In addisalinity . Ca is asalinity . Moreovesalinity . Salinitsalinity ,32; thussalinity ,34. Applsalinity . A numbeSimilarly, proline content in plant tissues is both a reflection and a measure of stress-induced damage at the cellular level. Accumulation of proline under stress protects the cell by balancing the osmotic strength of cytosol with that of the vacuole and external environment . Our res2O2, HO* and O2−) [The present study imitated an increase in activities of antioxidant enzymes  in response to NaCl-based osmotic stress due to the production of ROS (Hand O2−) . Our resand O2−) , who als+2  and aqueous Na2HPO4 (2 mM) in NaHCO3 (2 mM) were mixed drop-wise with a continuous stirrer at 37 °C and pH 7.4, until the appearance of suspended nanoparticles. The resulting ANPs were centrifuged, washed and resuspended in the nitrogen source (thiourea) for surface functionalization for 72 h under vigorous stirring. The resulting TU–ANPs were centrifuged at 8000 rpm, washed, freeze-dried and subjected to contemporary characterization, such as IR spectroscopy, SEM, EDX and XRD analysis using Oxford Inca 200 SEM instrument equipped with a Thermo EDX attachment and JEOL JDX 3532 X-ray diffractometer at the University of Peshawar.While following the biomimetic precipitation procedure of Paz et al.  with sliZea mays (SB-989 variety) in dH2O, 1 µg/mL, 5 µg/mL and 10 µg/mL of TU–ANPs were sown in Petri plates with double folds of Whatman filter paper under 0 mM, 60 mM and 100 mM of NaCl-induced osmotic stress with three replicates (30 seeds) in each treatment condition at 25 °C. The time to 50% germination (T50), mean germination time (MGT), percent germination (PG) and germination index were calculated using the Equations as follows:Ni is the number of seeds germinated on the day; (Ti) is the time (days); G1 is the germinated seeds on the first day (D1); Gf is the final germinated count on the final day (Df); N is the number of seeds germinated on the final day; Na and Nb are the respective cumulative germination at Ta and Tb (times in days); Gt is the total number of germinated seeds; and Nt is the total number of seeds.An experiment under laboratory conditions was performed using a randomized complete block design at the Department of Botany, University of Peshawar, Pakistan, to determine various germination indices. Primed seeds of Zea mays in primed in dH2O, 1 µg/mL, 5 µg/mL, and 10 µg/mL of TU–ANPs were sown in pots and filled with loam and ten seeds each at the Department of Botany, University of Peshawar, Pakistan . After completion of the seed germination (7 days), pots were placed under osmotic stress using 0 mM, 60 mM and 100 mM of NaCl solution. The stress was maintained with a weekly application of the mentioned doses of NaCl. Plants were, respectively, treated with dH2O, 1 µg/mL, 5 µg/mL and 10 µg/mL of TU–ANPs via the foliar route with an interval of 7 days for 6 weeks. Upon the completion of the seventh week, plants were harvested and initially subjected to data collection for vegetative growth parameters . Fresh plant specimens were also subjected to data collection in order to quantify photosynthetic pigments, osmolytes and antioxidant enzymes.Similarly, the field experiment was performed using a randomized complete block design. Seeds of v/v) of acetone, centrifuged at 8000 rpm. The OD of the supernatant was recorded at 645 nm, 663 nm and 450 nm. Similarly, the protein content in leaves was determined spectrophotometrically, following the procedure of Zhang et al., [Zea mays (500 mg) in PBS (pH 7.5) was mixed with alkaline Na2CO3 and Na-K tartrate (aqu.). To the reaction mixture, an aqueous CuSO4·5H2O solution and Folin phenol reagent was added and incubated for 30 min. The OD was recorded at 650 nm and the protein content was determined using the BSA standard curve.Photosynthetic pigments were quantified through a UV–Vis spectrophotometer according to Lichtenthaler and Wellburn  with sli et al.,  with som2O, centrifuged at 8000 rpm; to the supernatant, 80% of phenol and 5 mL of concentrated H2SO4 was added. The OD of the reaction mixture was recorded at 420 nm, and the total soluble sugar was estimated using a standard sugar curve. The amount of proline was determined by following the procedure given by Zhang [Sugar was extracted by homogenizing leaves in dHby Zhang . ProlineEnzymes were extracted from fresh leaves while homogenizing in 15 mL of 0.05N PBS (pH 7.0) with EDTA and PVPP. Homogenate was centrifuged at 80,000 rpm, and the supernatant was used to determine the activity of antioxidant enzymes.2O2 at 25 °C: the change in the OD was recorded at 290 nm for 3 min. The activity of ascorbic peroxidase was determined using the extinction coefficient of 2.8 mM cm−1 by calculating the amount of oxidized ascorbate. Catalase (CAT EC 1.11.1.6) was measured by adding enzyme extract to 2 mL of PBS (pH 7.4), 0.1 mL of EDTA and 20 mM of H2O2, and change in the OD of the reaction mixture was recorded at 240 nm for 3 min. The methodology of Wang et al. [2O2, and the change in OD value was measured at 485 nm for 20 min. In order to control for random errors, all of the experiments were replicated three times.For lipid peroxidase (EC 1.11.1.3), activity was determined using the TBARS assay. Enzyme extract was added to trichloroacetic acid and centrifuged at 4000 rpm. To the supernatant, 0.5% thiobarbituric acid in 2.5 N HCl was added in the water bath at 100 °C for about 30 min, allowed to cool in an ice bath. The OD was recorded at 532 nm and 600 nm against the blank-having reaction mixture, without leaves homogenizing. Ascorbic peroxidase (APX EC1.11.1.11) activity was determined, with some modifications, by following the method of Verma et al. . Enzyme g et al.  was follThe data was analyzed through a OW-ANOVA test for the comparison of multiple population means using the experimental sample data through the Statistix 10 package. All of the figures were plotted in Origin 9.1, where each of the mean values are from triplicate data with a 95% CI (error bars).Zea mays, whereas the application of TU–ANPs ameliorated plant growth via the conversation of photosynthetic pigments and production of osmolytes. The elevated activities of antioxidant enzymes, such as CAT, SOD, APX and POD, in plants under osmotic stress were also restored via foliar application of TU–ANPs. Seed priming and foliar application with optimal doses of TU–ANPs effectively elevated the tolerance level of the crop to osmotic stress and is therefore recommended.Calcium and phosphorus salts act as precursors for the biomimetic synthesis of apatite nanostructures. The thiourea acts as a surface functionalizing agent, validated through FTIR, X-ray diffraction spectroscopy, SEM and EDX spectroscopy. Osmotic stress delayed the germination and seedling growth of


Plasmodium falciparum. PfRipr5, a protein fragment of PfRipr inducing the most potent inhibitory antibodies, is a promising candidate for the development of next-generation malaria vaccines, requiring validation of its potential when formulated with adjuvants already approved for human use. In this study, PfRipr5 antigen was efficiently produced in a tank bioreactor using insect High Five cells and the baculovirus expression vector system; purified PfRipr5 was thermally stable in its monomeric form, had high purity and binding capacity to functional monoclonal anti-PfRipr antibody. The formulation of purified PfRipr5 with Alhydrogel®, GLA-SE or CAF®01 adjuvants accepted for human use showed acceptable compatibility. Rabbits immunized with these formulations induced comparable levels of anti-PfRipr5 antibodies, and significantly higher than the control group immunized with PfRipr5 alone. To investigate the efficacy of the antibodies, we used an in vitro parasite growth inhibition assay (GIA). The highest average GIA activity amongst all groups was attained with antibodies induced by immunization with PfRipr5 formulated with CAF®01. Overall, this study validates the potential of adjuvanted PfRipr5 as an asexual blood-stage malaria vaccine candidate, with PfRipr5/CAF®01 being a promising formulation for subsequent pre-clinical and clinical development.PfRipr is a highly conserved asexual-blood stage malaria vaccine candidate against  Plasmodium falciparum, namely, pre-erythrocytic vaccines, asexual blood-stage vaccines, and transmission-blocking vaccines. Last year, the World Health Organization (WHO) for the first time recommended the use of a pre-erythrocytic vaccine based on circumsporozoite protein (CSP), RTS,S/AS01, for the prevention of P. falciparum malaria in children living in regions with moderate to high transmission  using blood-stage parasites , and demonstrated the significant GIA activity of anti-PfRipr antibodies using both homologous P. falciparum 3D7 and heterologous FVO strains in vitro ] and PRIMVAC and PAMVAC  with a non-aluminum-based adjuvant, glucopyranosyl lipid adjuvant–stable emulsion (GLA-SE) were shown to be safe and well-tolerated, and induced higher levels of functional antibodies compared to aluminum-based adjuvant, Alhydrogel® ] stabilized with trehalose 6,6-dibehenate (TDB), a synthetic variant of mycobacterial glycolipid cord factor which is recognized by the C-type lectin receptor MINCLE and has been shown to be safe in human trials  and at 27°C, using shake-flasks  of 500 mL (N-4 stage) and 2000 mL (N-3 stage), and stirred tank-bioreactors (Sartorius) of 2 L (N-2 stage), 10 L (N-1 stage) and 50 L . For shake-flask cultures, cells were maintained in a shaking incubator  set to 100 rotations per minute (rpm) and with 2.54 cm shaking diameter. For bioreactor cultures, pO2 was set to 30% of air saturation and was maintained by varying the agitation rate from 60 to 270 rpm and the percentage of O2 in the gas mixture from 0 to 100%, the gas flow rate was set to 0.01 volume per volume per minute (vvm).PfRipr5 recombinant protein was produced in a 50 L stirred-tank bioreactor  by infecting insect High Five cells  at 2 ×10lsewhere . Cells wPurification of secreted PfRipr5 was carried out on ÄKTA Explorer 100 systems  as described elsewhere . In brieCell concentration and viability were assessed using a Cedex HiRes Analyzer .SDS-PAGE and Western blot analyses were performed as described elsewhere . ReducedProtein concentration was determined by spectrophotometry at 280 nm using the mySPEC equipment .The size distribution of the purified PfRipr5 was analyzed by dynamic light scattering (DLS) on a Spectro Light 600 .Purified PfRipr5 protein was analyzed in a HPLC system equipped with Photodiode Array Detector . Purified sample was loaded in a XBridge BEH 125 Å SEC 3.5 µm HPLC column (Waters), equilibrated in buffer containing 16 mM sodium phosphate, 250 mM NaCl, at pH 8.0, at a flow rate of 0.86 mL/min. Twenty micrograms of PfRipr5 was injected, and the eluted proteins were detected at 280 nm.Purified PfRipr5 was mixed with a thermal shift dye (Thermo Fisher Scientific) in a MicroAmp™ EnduraPlate™ Optical 96-Well Fast Clear Reaction Plate with Barcode (Thermo Fisher Scientific) to a final volume of 20 µL (n = 2 measurements). Thermal shift assay was performed in a QuantStudio 7 Flex RealTime PCR System (Thermo Fisher Scientific), with excitation and emission wavelengths of 580 and 623 nm, respectively. Plates were heated from 25°C to 90°C (rate of 0.016°C per second) and fluorescence was measured. Results were analyzed using the Protein Thermal Shift™ Software V1.3.Surface plasmon resonance (SPR) was carried out in Biacore X100 (Cytiva) as we previously described .® was provided from Croda Denmark , CAF®01 was provided from Statens Serum Institut , and GLA-SE was provided from Access to Advanced Health Institute . SSI and AAHI performed the compatibility studies using their routine assays to evaluate suitability of the adjuvants for formulation with PfRipr5. The PfRipr5 was diluted in 10 mM Tris buffer with 2% glycerol (pH=7.0) to the target concentration in each vaccine formulation. Five vaccine formulations (500 µL/dose) were devised, namely (i) Alhydrogel® vaccine formulations containing Alhydrogel® (5 mg/mL) and either 100 µg/mL (low dose) or 400 µg/mL (high dose) of PfRipr5, (ii) CAF®01 vaccine formulations containing CAF®01 (1250 µg/mL DDA and 250 µg/mL TDB), and either 100 µg/mL (low dose) or 400 µg/mL (high dose) of PfRipr5, and (iii) GLA-SE vaccine formulation containing GLA-SE (50 µg/mL) and 400 µg/mL (high dose) of PfRipr5. In human clinical trials, often 100 µg will be set as the highest dose. To evaluate a dose range, we used half (50 µg/dose) as low dose and twice (200 µg/dose) as high dose in this rabbit study. In the case of GLA-SE formulation study, if the higher antigen concentration formulation is compatible, we will be able to expect compatibility with the lower antigen concentration. Therefore, we only tested the high dose in the case of the GLA-SE formulation.The PfRipr5 antigen was formulated with three adjuvants compatible for human use. Alhydrogelvia visual inspection and pH measurement at room temperature (RT) to mimic the on-site preparation for Alhydrogel® or CAF®01 formulations one hour post formulation and for GLA-SE formulations at 0-, 4-, and 24-hour post formulation at RT and 5˚C. In addition, the interaction of the PfRipr5 antigen with Alhydrogel® or CAF®01 was further evaluated by mixing the adjuvant with 100 µg/mL or 400 µg/mL of the PfRipr5 protein, centrifuging at 14,000 ×g for 15 minutes (Alhydrogel® formulations) or 137,400 ×g for 30 minutes (CAF®01 formulations), and measuring non-adsorbed protein in the supernatant using bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). In the case of GLA-SE formulation, antigen stability was assessed by sandwich-ELISA established in our recent study using functional mouse anti-PfRipr mAb (clone 29B11) as capture antibody , and the antisera were provided by the company. In brief, Japanese white rabbits (n=6 per group) were subcutaneously immunized with the PfRipr5 protein alone (50 µg/shot) or with PfRipr5 antigen  formulated with the aforementioned adjuvants at the specific concentrations in 500 µL injection, twice at three-week intervals (Day 0 and Day 21). Antisera were collected two weeks after the last immunization (Day 35).ELISA was conducted to measure anti-PfRipr5 rabbit antibody titer. The following buffers were used: (i) coating buffer, containing 50 mM sodium carbonate buffer pH 9.5, (ii) blocking buffer, containing 1% (w/v) bovine serum albumin (BSA)  in phosphate buffered saline (PBS), (iii) dilution buffer, containing 0.1% BSA in PBS, and (iv) stopping buffer, containing 1 M sulfuric acid . Briefly, flat-bottom 96-well ELISA plates (Corning) were coated with 100 ng per well of PfRipr5 diluted with coating buffer. Plates were blocked with 300 μL/well of blocking buffer for 1 hour at 37˚ C. Five-times serial dilutions of each test rabbit serum starting from 1000-times dilution were prepared in dilution buffer. Diluted sera were added to antigen-coated wells in triplicate (50 μL/well) and incubated for 1 hour at 37°C. Plates were washed with washing buffer using a plate washer, and incubated with 100 μL/well of the goat anti-rabbit IgG conjugated with horseradish peroxidase  for 1 hour at 37°C . After washing, the substrate  diluted with 5 mM citric acid buffer pH 5.0 was added, and the plates were incubated at 37°C for 15 minutes. Reactions were stopped by adding 100 μL/well of stopping buffer. Absorbance was promptly measured at 492 nm using a Spectramax M3 microplate reader . Reciprocal serum dilutions that gave a mean absorbance value of 0.5 at 492 nm were determined as the endpoint titers.P. falciparum 3D7 strain for the GIA to determine the functional activity of anti-PfRipr5 IgG. P. falciparum 3D7 strain was kindly provided by the National Institute of Allergy and Infectious Diseases (NIAID), and asexual stage of the parasite was cultured as described elsewhere , airtight boxes. After 25 hours of incubation, when most of the invading parasites had developed to the early trophozoite stage, the pRBC were pelleted by brief centrifugation  and washed once in 100 µl PBS. The cells were then incubated with 50 µl of diluted  SYBR green I (Invitrogen) for 10 min at RT and washed once in PBS. Parasitemia was measured by flow cytometry with a FACSCanto II  by the acquisition of 50,000 events per sample. Data were analyzed with FlowJo 9.1 software  by first gating for intact erythrocytes by side scatter and forward scatter parameters and subsequently determining the proportion of SYBR green I-positive cells. Rabbit IgGs obtained after immunization with Freund adjuvant formulated hexa-histidine-tagged glutathione S-transferase (His-GST) and region 3 to 5 of erythrocyte binding antigen 175 of P. falciparum (PfEBA175)   were useAll statistical analyses were performed with GraphPad Prism (ver. 9.4.0) . Difference of the mean antibody titers and GIA activities among groups was tested by one-way ANOVA with Tukey’s multiple comparisons test. Pearson’s correlation coefficient between ELISA titers and GIA activities was calculated. P-values less than 0.05 were considered as statistically significant.PfRipr5 was produced using insect High Five cells and the baculovirus expression vector system (IC-BEVS) at 50 L scale, and the quality of purified product was assessed by SDS-PAGE, western blot, Dynamic light scattering (DLS), High performance liquid chromatography-Size-exclusion chromatography (HPLC-SEC), Thermal shift assay (TSA) and Surface plasmon resonance (SPR).. In addition, the thermal stability of purified PfRipr5  as well as its ability to bind to the anti-PfRipr mAb 29B11  were confirmed , onset of cell viability drop at 48 hpi, and culture harvest at 72 hpi (when cell viability reaches approx. 80%) . Bands c. In addiThese data demonstrates that the purified PfRipr5 antigen herein produced in insect cells had high quality and thus was suitable for further formulation with adjuvants and animal immunizations.®, CAF®01, and GLA-SE, and the PfRipr5-adjuvant compatibility was confirmed following analyses optimized for each adjuvant.The PfRipr5 antigen was formulated with three different adjuvants, Alhydrogel® or CAF®01 showed no changes as compared to Alhydrogel® or CAF®01 alone, respectively. Likewise, the pH of the PfRipr5/Alhydrogel® (7.2) and the PfRipr5/CAF®01 (7.0) was similar to that of each adjuvant alone, thus suggesting their chemical stability  than all the adjuvant alone groups (Alhydrogel®(Alum), GLA-SE (GLA), and CAF®01 (CAF) with 0 µg PfRipr5, P<0.05) suggesting that the PfRipr5 protein itself is immunogenic in rabbits  (Mean ELISA titers: Alum = 1.0 ×105 (P<0.01); CAF = 1.0 ×105 (P<0.01)) and in all high dose groups (200 µg) than the PfRipr5 alone group, the exception being the low dose (50 µg) formulation with GLA-SE (Mean ELISA titer = 8.0 ×104); no statistically significant difference was attained for the anti-PfRipr5 antibody titers between high and low dose within the adjuvant groups and across the adjuvant groups.Rabbits (n=6 per group) were subcutaneously immunized with the above-mentioned formulations. PfRipr5 alone (50 µg) induced significantly higher anti-PrRipr5 antibodies (Mean ELISA titer = 3.4 ×10in vitro GIA was conducted. The GIA activities of IgG induced by PfRipr5 GLA-SE and CAF®01 formulations were higher in the high dose (200 µg)  than in the low dose (50 µg)  groups, respectively (® formulation was higher in the low dose (50 µg) (Mean %GIA = 37%) than in the high dose (200 µg) (Mean %GIA = 19.9%) groups. In ® formulation (P<0.01), high dose (200 µg) PfRipr5/GLA-SE (P<0.01), and low (50 µg)(P<0.01) and high dose (200 µg)(P<0.0001) PfRipr5/CAF®01 groups showed statistically significant levels of GIA activities compared to the PfRipr5 (50 µg)/no-adjuvant group. In addition, significantly higher GIA activity was also observed in the high dose (200 µg) PfRipr5/CAF®01 than low dose (50 µg) PfRipr5/GLA-SE groups. No statistically significant difference was attained for the other GIA activities between high and low dose within the adjuvant groups and across the adjuvant groups. However, importantly, eight rabbit IgG samples out of 60 samples demonstrated ≧50% GIA activities, belonging to the PfRipr5/CAF®01 groups  and high dose PfRipr5/GLA-SE (one rabbit). Notably, if one outlier rabbit in the high dose (200 µg) PfRipr5/CAF®01 group was excluded from the analysis  PfRipr5/GLA-SE group with similar levels of anti-PfRipr5 antibody titers, although statistical difference has not been reached significant. This observation indicates that higher IgG titer correlates with higher GIA activity and suggests that further efforts should focus on how to increase the anti-PfRipr5 specific antibody titers for the development of a potent PfRipr5-based vaccine formulation.Anti-PfRipr5 antibody titers and GIA activities from 60 rabbits showed statistically significant positive correlation  , to prioritize a PfRipr5-based malaria vaccine candidate for further advance its development. The PfRipr5 antigen herein produced was shown to be thermally stable in its monomeric form, having high purity and binding capacity to functional monoclonal anti-PfRipr antibody, thus assuring its quality for the subsequent use in animal experiments.In this study, we tested head-to-head the adjuvanticity of PfRipr5 protein formulated with three adjuvants for human use  . Finally, higher mean GIA activity was also observed in the high dose CAF®01 formulation than high dose GLA-SE group even with similar levels of anti-PfRipr5 antibody titers  group was the highest among all the groups (approximately 50% inhibition), which is similar to the GIA activity of antibodies elicited against PfRipr5 with non-human applicable Freund’s adjuvant formulation . All animal immunizations were commercially conducted at Kitayama Labes Co., Ltd. .ET, AR, AF, NV, HD and TT conceived and designed experiments. ET, HN, RC, DC, JG and AR conducted experiments. ET, AF, PA, AR and TT analyzed the data. ET, AF, NV, RC, HD and TT wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was funded by the Japan-based Global Health Innovative Technology (GHIT) Fund (grant ID: T2018-151). This work was also supported in-part by JSPS KAKENHI  and by a research fund from Sumitomo Pharma Co., Ltd. iNOVA4Health – UIDB/04462/2020 and UIDP/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior, through national funds is acknowledged. Funding from INTERFACE Programme, through the Innovation, Technology and Circular Economy Fund (FITEC), is gratefully acknowledged. This work was supported by FCT through the initiatives “Investigador FCT” Program (IF/01704/2014), Exploratory Research and Development Project (IF/01704/2014/CP1229/CT0001), and PhD fellowship (Ricardo Correia - SFRH/BD/134107/2017). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.® was a kind gift from Dr. Erik B. Lindblad, R&D Unit, Croda, Denmark. We thank the Japanese Red Cross Society for providing human erythrocytes and plasma for culturing P. falciparum parasites. We also thank Miwa Ochi, Yuiko Ogasawara, Ai Yanase, and Yusuke Hara for their technical assistance, and Flavia D’Alessio, Ezenwa James Onyemata, Robert Adamu Shey and Masauso Moses Phiri for project management support.The AlhydrogelAF is employed by Sumitomo Pharma Co., Ltd. and TT and ET are supported by a research fund from Sumitomo Pharma Co., Ltd. AF, TT, ET, and HN are inventors on patent PCT/JP 2017/040532 on a malaria vaccine antigen, PfRipr5. DC is co-inventor on patents on the cationic adjuvant formulations CAF. All rights have been turned over to SSI, which is a non-profit government research facility. JAG works for AAHI which has patent rights in the GLA-SE adjuvant formulation. These involvements did not influence the design of the study, the collection, analysis, access to, and interpretation of data, or the writing of the manuscript.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Patients with controlled acromegaly had lower fasting glucose  and were less likely to have persistent T2D  and HBP . There were strong positive correlations between BIA and DXA for fat mass  and muscle mass parameters: SMI X Baumgartner: r=0.890, p<0.001; and FFMI X Baumgartner: r=0.868, p<0.001.Although patients with controlled disease had lower serum GH (1.2 ± 1.68µg/L our data showed similar results in body composition assessment by BIA and DXA, with good correlation between the methods, regardless of the hormonal status of acromegaly. Furthermore, in patients with adequate hormonal control, there was preservation of muscle mass and a lower prevalence of metabolic comorbidities, such as T2D and HBP. Acromegaly is a rare and slowly progressive disease , charactBy obtaining hormonal control with normalization of serum IGF-1 for age and GH below 1µg/L , 11, theThe few data available on body composition in patients with acromegaly have yielded conflicting results –18. SomeThe present study aims to evaluate body composition in patients with acromegaly, using both dual X-ray absorptiometry (DXA) and multifrequency bioelectrical impedance analysis (BIA), which is portable, cheaper and widely available compared to DXA, and its relationship with hormonal control and metabolic parameters.We studied 28 consecutive patients (22 women and 6 men), with a mean age of 52.11 ± 12.53 years and disease duration of 6.43 ± 4.68 years (1 to 20 years). The diagnosis of acromegaly was confirmed by an increase in serum IGF-1 for age, associated with serum GH nadir during an 75g-oral glucose tolerance test (OGTT) above 1µg/L (or above 0.4µg/L for ultrasensitive assays), in the presence of characteristic clinical features , active cancer , pregnant women, acute diseases (coronary syndrome or liver failure) as well as chronic renal failure with a glomerular filtration rate below 30 mL/min were excluded.After signing the informed consent, the patients answered a specific clinical questionnaire and underwent a complete clinical examination; after overnight fasting, blood was collected for laboratory tests and body composition analysis was performed (BIA and DXA), with no database information being used. Serum GH was measured by chemiluminescence using Access UniCel DXI 800 , with inter- and intra-assay coefficients of variation < 6%. Serum IGF-1 was measured by chemiluminescence , with intra- and inter-assay coefficients of variation of 4% and 5.9%, respectively. Serum testosterone and estradiol were also measured by Immulite 2000. Fasting plasma glucose (FPG), glycated hemoglobin (HbA1c), total cholesterol (TC), HDL cholesterol, LDL cholesterol and triglyceride were measured using an auto-analyser .2) and skeletal muscle mass index ; and by DXA: Baumgartner index  and Foundation for the National Institutes of Health [FNIH] (appendicular muscle mass divided by BMI). Body fat by both BIA and DXA was expressed as percent fat in relation to body weight. Pituitary imaging was performed by gadolinium-enhanced magnetic resonance .Body composition analysis was performed by Multifrequency (8-electrode) Bioelectrical impedance (BIA)  and total body densitometry (DXA) . The following parameters were used by BIA: fat-free mass index .Data were reported using absolute frequencies and percentages for categorical variables and mean and standard deviation for numerical variables. Correlation between two numerical variables was evaluated by Spearman’s correlation coefficient and the corresponding Student’s t-test for the null correlation hypothesis. The comparison between two categories in relation to numerical variables was performed using one of the tests: t-Student with equal variances, t-Student with unequal variances or the Mann-Whitney test. Statistical significance was considered if p<0.05. Data were plotted in Excel spreadsheet and the software used to obtain statistical calculations was the IMB SPSS version 25.Of the 28 patients, 23 underwent surgery, with 4 patients cured postoperatively. The remaining 24 patients were using adjuvant drug therapy and 4 underwent radiotherapy (3 remained with active disease). Patients were divided into two groups according to hormonal control  , 11: actvs 6.69 ± 5.75 years, p = 0.787, respectively for inactive and active groups. Patients with active disease have remained on adjuvant treatment since diagnosis, while the group with controlled disease (inactive) has had adequate hormonal levels for the last 2,8 years .Regarding the treatment of acromegaly, 24 patients used adjuvant medical therapy: 21 patients were on cabergoline (CAB) plus first-generation somatostatin receptor ligands (SRL) . Three patients used exclusively CAB with a median dose of 2mg/week (0.5 to 3.5mg/week). All patients received at least one type of treatment  and none of the patients used pegvisomant or pasireotide. The groups did not differ in terms of diagnosis time: 6.2 ± 3.71 years vs 1.2 ± 1.68µg/L, p=0.001) and IGF-1 concentrations , larger pituitary tumors both at diagnosis  and at study entry  (Patients with active disease had significantly higher serum GH compared to inactive disease (6.61 ± 6.40µg/L p=0.053) Figure 1vs 37.73 ± 8.76%, p=0.691 for BIA and 37.10 ± 10.21% vs 37.89 ± 7.32%, p=0.819 for DXA), FFMI , Baumgartner  and FNIH , respectively . Similarly, 71.4% of patients with active disease had T2D while 28.6% of patients with inactive disease had persistent T2D (p=0.008) Figure 2There were strong positive correlations between BIA and DXA for fat mass  and muscle mass parameters: SMI-BIA X Baumgartner-DXA: r=0.890, p<0.001; and FFMI-BIA X Baumgartner-DXA: r=0.868, p<0.001 Figure 3In the present study we demonstrated preservation of muscle mass despite a fall in serum GH/IGF-1 concentrations with disease control in patients with acromegaly. Few studies have addressed changes in body composition in acromegaly and have prioritized the assessment of fat mass, mostly suggesting its increase after obtaining hormonal control , 15, difDifferent technologies are available for quantification of fat and muscle mass –23. AlthCorrelation studies between BIA and DXA have already been performed in other conditions such as primary hyperparathyroidism  and follvs acromegaly with GH sufficiency 54.1 ± 2.7kg vs acromegaly with GH deficiency 58.5 ± 2.7kg, p = 0.11). These findings may be related to the long period of exposure to excess GH, inducing persistent structural skeletal muscle changes, that led to a non-significant loss of lean mass after hormonal control of acromegaly . The patients/participants provided their written informed consent to participate in this study.AAL Data Collection, Data analysis, Manuscript writing and review, Manuscript submission; LA: Data analysis, Manuscript writing; MF: Data Collection, Data analysis; DR: Data Collection, Data analysis, Manuscript writing; FB: Project development, Manuscript writing, editing and review. All authors contributed to the article and approved the submitted version.This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES – Finance Code 001).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We could show hypo-reactive responses of T lymphocytes isolated from knockout mice with a known defect in T lymphocyte activation. Thus, this MLR-based in vivo model provides the opportunity to analyze positive regulators of T cell responses under physiological conditions of polyclonal T lymphocyte activation in vivo.Depending on the context, robust and durable T lymphocyte activation is either desirable, as in the case of anti-tumor responses, or unwanted, in cases of autoimmunity when chronic stimulation leads to self-tissue damage. Therefore, reliable in vivo models are of great importance to identify and validate regulatory pathways of T lymphocyte activation. Here, we describe an in vivo mixed-lymphocyte-reaction (MLR) approach, which is based on the so-called parent-into-F1 (P → F1) mouse model in combination with the congenic marker CD45.1/2 and cell proliferation dye-labeling. This setup allows us to track adoptively transferred allogenic CD4 The importance of a finely balanced T cell response—strong enough to prevent cancer and to protect from infectious diseases while not damaging its own tissue—is reflected in the increasing amount of therapeutic approaches targeting T cells. These strategies aim to either suppress T cell activity, as in the case of autoimmunity and organ transplantation, or boost it, which is preferable for anti-tumor approaches. The latter applies, for example, for an adoptive transfer of T cells (ACT) expressing a chimeric antigen receptor as a therapeutic tool. In this regard, it is of great interest to understand the molecular switches of T cell activation, to modulate their responsiveness and the T cell-mediated immune response outcome. Thus, there is a strong need for physiological model systems to test potential modulators of T cell responses.6–108, [However, since T cells demonstrate an enormous range . However, the tracking of T cell activation using this in vivo approach is challenging due to the low frequency of (naïve) antigen-specific precursors . Thus, + and CD4+ T cells, respectively, harbor a TCR specific for a peptide from chicken ovalbumin [+ T cells due to MHC class I-mediated positive selection. Furthermore, no polyclonal response is analyzed using this approach.Other in vivo models are based on an ACT of TCR-transgenic (tgTCR) T cells, using a monoclonal TCR, followed by immunization with the cognate antigen. For instances, OT-I and OT-II mice are used, whose CD8valbumin ,8. In thAnother possibility to address T cell responses in vivo is based on the above-described MLR approach, which increases the frequency of potential responders to approximately 7% . TherebyUnlike the aGVHD model described above, the so-called parent-into-F1 (P → F1) model avoids rejection of the transferred T cells of a certain haplotype by using semi-allogenic F1-hybrids of this haplotype and of an allogenic haplotype as a host. For instance, C57BL/6 splenocytes or sorted T cells (MHC haplotype b) are transferred to offspring of a C57BL/6 × DBA2 breeding (b/d haplotype). Using this approach, the irradiation of the recipients is not necessary. The transferred parental strain T cells will respond vigorously to the mismatched MHC d haplotype of the recipient.PKCθ-knockout mice. PKCθ is a central signaling molecule downstream of the TCR, activated by DAG-binding, whose deficiency is known to impair T cell activation resulting in reduced interleukin (IL)-2 expression and proliferation . Thu. ThuPKCθ6 MACS-sorted CD3+ T cells from CD45.2+/2+ C57BL/6 mice, labeled with the proliferation dye eFluor670, were adoptively transferred by intravenous injection into syn- or allogenic recipients  to CD45.2+/2+ DBA2 males (MHC haplotype d), resulting in CD45.1+/2+ B6D2F1 hybrids (MHC haplotype b/d). Syngenic CD45.1+/2+ C57BL/6 mice (haplotype b) were used as control recipients.In order to investigate T cell activation upon allogenic stimulation, we have adapted the P → F1 model by the additional inclusion of a congenic marker. In brief, 8 × 10cipients A. The laBased on the expression of the congenic marker CD45, the transferred T cells can be now identified by flow cytometry. Proliferation was analyzed by determining the dilution of the eFluor670 dye, which binds irreversibly to intracellular proteins and is equally distributed between two daughter cells. The analyses were performed on day 3, a rather early time point upon ACT, and at day 8 as a later time point; however, this was before any GVHD symptoms should occur . Indeed,+ T cells responded faster than CD8+ T cells, with more divided CD4+ than CD8+ T cells at day 3 after ACT, which is in accordance with the literature on the P → F1 model, describing that donor CD4+ T cells initiate the GVHD response [+ T cells catch up, and at day 8 after ACT they had undergone at least eight cell cycles so that “all proliferation dye label” has been lost, now showing the same fluorescence intensity as unlabeled cells. The different proliferation kinetic of CD4+ and CD8+ T cells is also evident by the CD4+ to CD8+ T cell ratio. Thus, CD4+ T cells reach a 5.5 ± 0.3 fold number of CD8+ T cells at day 3, but the ratio declines on average below one (0.9 ± 0.1) at day 8. Accordingly, T cell differentiation and acquisition of an effector/memory phenotype, visible by the loss of CD62L expression and upregulation of CD44, was observed in allogenic but not syngenic recipients -deficient mice, a knockout-mouse strain known to have an impaired T cell response, were used. Others, as well as our laboratory, have already demonstrated that PKCθ is activated downstream of the TCR complex and is involved in early T cell activation ,15. Thus+ T cells) was quite similar to wild type T cells. However, the frequency of divided T cells lacking PKCθ in contrast to wild type T cells did not further increase with time, but rather declined , the activation and proliferation of CD4+ T cells was similar between the four distinct groups, 6 transferred T cells are enough to analyze in vivo activation in this allogenic model.To enable analysis in case less T cells  are available for ACT, and also to reduce the number of needed donor mice, we tested four different numbers of transferred T cells  and analyzed recipient mice at day 8. The spleen weight was comparable between 1, 2 and 4 million transferred T cells, and only significantly increased when 8 million T cells were transferred A. Additi+ T cells expand and provide help for host B cells, which proliferate and produce autoantibodies. In addition, donor CD8+ T cells will be activated and differentiate into cytotoxic T lymphocytes (CTL) that eliminate host lymphocytes, primarily B cells. The latter response leads to aGVHD with a profound immunodeficiency by day 14, counteracting donor-CD4+/host-B cell-mediated autoimmune symptoms . Of notescribed . Further T cells . Altogetof aGVHD . Hence, + T cell expansion and maturation, thereby attenuating aGVHD symptoms in the B6 → F1 model [Of note, using the P → F1 model, different approaches have been conducted to modulate T cell responses and thereby affect the disease outcome to induce either acute or chronic GVHD. In brief, while the blocking of CTLA-4 simultaneous with ACT prevented the development of acute and chronic GVHD, other treatments promoted either one or the other disease phenotype . TreatmeF1 model ,25,26.6 C67BL/6 donor T cells are used [The mentioned findings focus primarily on the outcome of the two major P → F1 disease phenotypes, which can be mainly distinguished by the extent of donor T cell engraftment, elimination or expansion of host B cells and serum anti-DNA antibody levels, at later time points (10 days to months). However, we are interested in rather early events of T cell activation on a cellular and molecular level. Thus, we modified the P → F1 model to assess how genetic alterations influence T cell activation by analyzing the phenotype of T cells from knockout mice within 8 days after transfer. Using this time frame for our analyses, no massive killing of endogenous cells and also no severe health issues are expected from what is known from earlier studies . This moare used ). Furtheare used ,28. BesiWe believe that this model based on transferring congenic allo-reactive T cells is a valuable tool for the investigation of genes of interest regarding a physiological T cell activation and effector function in vivo. In addition to the here described readouts, it is also possible to investigate tissue localization of the transferred T cells, for example by immunohistochemistry, and tissue infiltrating cells, e.g., from the gut, could be analyzed by flow cytometry.+ as well as CD8+ T cells in allogenic but not syngenic recipients. In this regard, the flow cytometric analysis revealed that the donor T cell phenotype changes from a dominantly naive to an exclusively effector/memory state and acquires exhausted profiles on day 8 after ACT. This can also be observed in a blood kinetic over time  with the accumulation of proliferated cells and a switch from a naive to an effector phenotype. However, this was all impaired in the case of PKCθ-deficient donor T cells, supporting our attempt to use this model in the future to determine genes that might be positive regulators of T cell functions. Our results, demonstrating reduced allogenic proliferation of T cells lacking PKCθ, are in line with what is known from the literature implicating a role of PKCθ in alloreactivity and GVHD. Thus, PKCθ-deficient donor T cells demonstrated reduced ability to induce disease in MHC-mismatched hosts  does not induce aGVHD [+ or CD8+ T cells separately or performing a transfer of genetically modified CD4+ T cells together with wild type CD8+ T cells, or the other way around, depending on the research question.Furthermore, we could demonstrate that downscaling the number of transferred T cells is possible. In this case, it is feasible to use the model if less cells are available; we tested as few as one million transferred T cells. However, one has to be aware that decreasing numbers of transferred T cells might impede the analysis. In particular, the tracking of parameters over time in blood samples will be difficult, since there is only limited availability of blood, which can be harvested per time point from a living animal. Moreover, we observed that CD8ce aGVHD . NeverthTaken together, our findings prove that the here described model is well suited to investigate regulators of T cell responses at a single cell level in vivo. Here, we focused on a basic setting, where genetically modified T cells are transferred to allogenic recipients serving as a starting point for a highly flexible, physiological model system investigating T cell activation in vivo, which can be adapted to one’s specific research questions and technical possibilities.+/1+”) females to receive a hybrid . Syngenic controls were obtained by the breeding of C57BL/6J (strain #000664) to B6.SJL-Ptprca Pepcb/BoyJ mice. PKCθ-deficient mice  and crossed with B6.SJL-Ptprca Pepcb/BoyJ  for 2-6 min at room temperature (RT), followed by a wash/ filter step using 1× PBS, 0.5% BSA, 0.5 M EDTA and 40 µM cell strainers . Viable cell counts were determined after staining with acridine orange and propidium iodide (AO/PI) Cell Viability Kit  on a LUNA Automated Cell Counter .Single cell suspensions of spleens were prepared by mechanical disintegration using metal sieves or 100 µM cell strainers . Thereafter, erythrocytes were removed by lysis  along with pre-separation filters, LS columns and a QuadroMACS separator , according to the manufacturer’s instructions. Sort purity was checked by flow cytometry.CD3+ T cells were labeled with cell proliferation dye eFluor670 (ebiosciences 65-0840). Therefore, a twofold labeling solution (10 µM) was prepared in HBSS with Mg2+ and Ca2+ (HBSS++). T cells were washed twice with HBSS++ at RT and adjusted to 1 × 107 cells/mL. An equal volume of the twofold labeling solution was added and mixed immediately with the cells by inverting the tube. Cells were incubated for 5 min at 37 °C in the dark. The reaction was stopped with cold FCS and RPMI followed by a wash step with RPMI and subsequently with HBSS++. Finally, cells were resuspended in HBSS++ (8 × 106/100 µL) and injected intravenously.Isolated CD3+ T cells from C57BL/6 mice  were isolated as described above, labeled with proliferation dye and transferred to allogenic B6D2F1 (MHC haplotype b/d) or syngeneic control (MHC haplotype b) recipients. A total of 1, 2, 4 or 8 × 106 T cells resuspended in 100 µL HBSS++ were injected into the lateral tail vein. For endpoint analyses, spleens from recipient mice were harvested and weighted on day 3 or day 8 after ACT. Total splenocyte counts were determined from single cell suspension after the lysis of erythrocytes. For T cell analyses from blood, the mice were bled from the facial vein using a lancet into tubes containing heparin. Subsequently, splenocytes or blood samples (after lysis of erythrocytes) were analyzed by flow cytometry.CD3Flow cytometric analyses were performed on FACS Canto II  and the subsequent data processing was conducted with the FlowJo software 10.8.0 . For surface stainings, the cells were incubated for 5 min with FcR-block  prior to the addition of the antibody mix in PBS, 0.5% BSA, 0.5 M EDTA. Subsequently, the cells were incubated for 20 min with the antibody solution. Lastly, the cells were washed with PBS, 0.5% BSA, 0.5 M EDTA and transferred into FACS tubes . The following antibodies were used for surface staining: CD4 V500 , CD8 PerCPCy5.5, CD8 BV510, CD8 APC-Cy7 , CD44 PE-Cy7, CD44 FITC , CD45.1 PB, CD45.1 APC-Cy7 , CD45.2 FITC, CD45.2 PE-Cy7 , CD45.2 V500  and CD62L APC-Cy7 . To exclude dead cells, the cells were stained with the Fixable Viability Stain 780  for 10 min at RT in the dark according to the manufacturer’s instructions. The following gating strategy was applied: (1) exclusion of dead cells via viability staining, (2) gating on lymphocytes in the FSC/SSC plot, (3) double cell exclusion, (4) gating on transferred cells via CD45.1/2 and (5) CD4 or CD8 blotted against proliferation dye and (6) gating on the parameter of interest .t-test (for simple comparisons between two groups), as indicated in the figure legends. A p-value of <0.05 was considered statistically significant. Symbols used in the figures are: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.Data were analyzed using the GraphPad Prism 9.0.1 software . The data were analyzed for statistical significance by one-way ANOVA following Bonferroni’s post hoc test (for comparisons on more time points or more groups) or by the Student’s


The cell cycle was studied by flow cytometry, mRNA and protein levels were analyzed by RT-qPCR and Western blot, respectively, and finally, cytoskeleton remodeling and NF-κB translocation were determined by confocal microscopy. Genistein 100 µM decreased cell viability and produced G2/M arrest, increased H2O2, and produced filopodia in SW620 cells. In HT29 cells, genistein produced an increase of cell death, H2O2 production, and in the number of stress fibers. In HT29 cells, mitochondrial biogenesis was increased, however, in SW620 cells, it was decreased. Finally, the expression of inflammation-related genes increased in both cell lines, being greater in SW620 cells, where NF-κB translocation to the nucleus was higher. These results indicate that high concentrations of genistein could increase oxidative stress and inflammation in colon cancer cells and, ultimately, decrease cell viability.Genistein could play a crucial role in modulating three closely linked physiological processes altered during cancer: oxidative stress, mitochondrial biogenesis, and inflammation. However, genistein’s role in colorectal cancer remains unclear. We aimed to determine genistein’s effects in two colon cancer cells: HT29 and SW620, primary and metastatic cancer cells, respectively. After genistein treatment for 48 h, cell viability and hydrogen peroxide (H Genistein (GEN) is a phytoestrogen that belongs to the isoflavones class and is found in soybeans . Asian cThe chemopreventive activity of GEN can modulate different physiological processes during cancer, such as oxidative stress , cancer GEN has a dual effect in front of oxidative stress. It is well known that GEN acts as an antioxidant during oxidative stress, increasing antioxidant enzymes’ expression , but GENROS production is closely related to mitochondrial biogenesis, which is a complex process where the nuclear and mitochondrial genome must be highly regulated, since the nuclear genome codifies proteins which control the transcription and replication of mtDNA that, in turn, codifies some mitochondrial proteins such as electron transport chain proteins . This prIn the same way, colorectal tumors suffer an increase of proinflammatory cytokines’ expression and a constitutive activation of transcription factors related to inflammatory pathways . These cOur aim was to investigate the effects of genistein treatment in oxidative stress, mitochondrial biogenesis, and inflammatory parameters in two colon cancer cell lines: HT29, a primary and moderately differentiated cell line, and SW620, a metastatic and poorly differentiated cell line. For this goal, cell viability, ROS production, the cellular cycle, gene and/or protein expression levels of antioxidant enzymes, mitochondrial biogenesis regulators and inflammation-related genes, the nuclear translocation of nuclear factor kappa B (NF-κB), and actin cytoskeleton remodeling were studied.Cell survival is key in cancer progression; consequently, the effect of GEN in cell viability was studied. 0/G1 phase (−52%), but suffered an increase in the Sub G0/G1, S and G2/M phases . SW620 cells  and an increase in the Sub G0/G1 and G2/M phases .The cell cycle  was stud20 cells B present2O2 production was analyzed after 48 h of GEN treatment , without significant changes with the lower concentrations.To know the origin of cell death, the oxidative stress status was studied. The Hreatment . HT29 ce2O2 production, the mRNA expression levels of different antioxidant enzymes were studied after 48 h of GEN 100 µM treatment. HT29 cells (SOD2 (+40%) and SOD1 (+36%) mRNA expression levels after GEN treatment. Moreover, in SW620 cells (SOD2 (+96%) and GPX1 (+17%), in addition to a statistically significant decreased expression level of CAT (−28%). Furthermore, the changes in the SOD2/CAT , as well as SW620 cells .To understand the changes in H29 cells A suffere20 cells B, GEN trSOD2/CAT C and SODOD2/GPX1 D ratios SW620 cell line gene expression changes were more pronounced than in HT29 cells. In consequence, the protein expression levels of the same antioxidant enzymes were studied in both cell lines after 48 h of GEN 100 µM treatment. HT29 cells A showed Actin cytoskeleton remodeling was studied with phalloidin staining after 48 h of GEN 100 μM treatment. PPARGC1α and ESRRA  after GEN treatment, while TFAM and SSBP1 suffered a statistically significant increase . In contrast, SW620 cells . Furthermore, mitochondrial DNA expression levels  and a statistically significant decrease in CXCL8 (−19%) mRNA expression after GEN treatment. On the other hand, SW620 cells  and a statistically significant decrease in PPARG (−70%) mRNA expression after GEN treatment. IL1B in SW620 cells also suffered an increase since the control cells showed a 40.6 ± 3.1 crossing point and GEN-treated cells showed a 31.9 ± 0.2 crossing point (data not shown).To understand the changes in inflammation, the mRNA expression levels of different interleukins and their receptors, as well as key genes of different inflammation-related pathways, were studied after 48 h of GEN 100 µM treatment. The HT29 cell line A present20 cells B suffereInflammatory-related gene changes were more pronounced in the SW620 cell line than in the HT29 cell line, thus, in order to have a more functional parameter related to inflammation, NF-κB translocation to the nucleus was studied after 48 h of GEN 100 µM treatment. The effects of high concentrations of the phytoestrogen GEN (100 µM) in colon cancer cells were analyzed to see the action of this phytoestrogen in cell viability, oxidative stress, mitochondrial biogenesis, and inflammation in colorectal cancer.Some studies about GEN metabolization and digestion suggest that it could be accumulated in various colon areas, where GEN may exert its effects . For thi0/G1 phase was previously seen by Salti and collaborators in the HT29 cells with a treatment with GEN 100 µM for 72 h  (ATCC CCL-227); Manassas, VA, USA), and cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) (2O2) production determinations. For further experiments, both cell lines were treated only with 100 μM of GEN for 48 h, and control vehicle-treated cells were treated with 0.1% DMSO.Cells were seeded at similar passages and treated, the following day, at 70% of confluency with increasing concentrations  of GEN with 0.1% dimethyl sulfoxide (DMSO) as the vehicle, and the control vehicle-cells were treated with 0.1% DMSO for 48 h for cell viability and hydrogen peroxide , which emits blue fluorescence when it is bound to double strand DNA. After GEN treatment, the culture medium was removed, and 5 μg/mL of Hoechst was incubated for 5 min at 37 °C. Finally, FLx800 microplate fluorescence reader  was used to measure the fluorescence, set at excitation wavelength of 350 nm and emission wavelength of 455 nm.Total of 5 × 102O2 production was determined using an Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit , according to the method described by Pons et al. [4 HT29 cells and 4 × 104 SW620 cells were seeded in each well in a 96-well plate and treated with increasing concentrations of GEN for 48 h. FLx800 microplate fluorescence reader  was used to measure fluorescence, set at excitation wavelength of 570 nm and emission wavelength of 585 nm. The maximum slope of the increase in the fluorescence was detected within 30 min of exposure to kit reagents. The number of viable cells determined by Hoechst 33342, as previously described, was used to normalize the obtained values.Hs et al. . Total o6 HT29 cells and 2.1 × 106 SW620 cells were seeded in each well in 6-well plates and treated with GEN 100 µM for 48 h. Then, total RNA was isolated by using Tri Reagent®  following the manufacturer’s protocol. A BioSpec-nano spectrophotometer  was used to quantify the total RNA amount, set at wavelength of 260 nm. The RNA quality was checked by 260/280 and 260/230 ratios.Total of 1.5 × 10Then, 1 μg of the total RNA was reverse transcribed to cDNA, according to Pons et al., . cDNA alSOD1), manganese superoxide dismutase (SOD2), glutathione peroxidase (GPX1), catalase (CAT), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1α), estrogen-related receptor alpha (ESRRA), transcription factor A (TFAM), single-stranded mitochondrial binding protein 1 (SSBP1), tumor necrosis factor alpha (TNF), interleukin-1 beta (IL1B), interleukin-8 (CXCL8), interlukin-8 receptor (CXCR2), heparanase (HPSE), interlukin-10 (IL10), and peroxisome proliferator-activated receptor gamma (PPARG) genes were analyzed, using the expression of beta-2-microglobulin (B2M) as housekeeping gene. Genes, primers, and annealing temperatures used are shown in A LightCycler 480 System II rapid thermal cycler  with SYBR Green technology was used to carry out the real-time PCR. The expression of copper–zinc superoxide dismutase  was used considering the efficiency of the reaction for each pair of primers and normalizing with the 6 HT29 cells and 2.1 × 106 SW620 cells were seeded in 6-well plates and treated with GEN 100 µM for 48 h. Then, DNA was isolated by using Tri Reagent® , following the manufacturer’s protocol. A BIO-TEK PowerWave XS spectrophotometer was used to quantify the DNA amount, set at wavelength of 260 nm. The DNA quality was checked by 260/280 ratio. A total amount of 5 ng of DNA was carried out into a real-time PCR, as previously described, and the expression of mitochondrial DNA (mtDNA) was analyzed, using the expression of 18S as housekeeping gene. Genes, primers, and annealing temperatures used are shown in Total of 1.5 × 105 HT29 cells and 8 × 105 SW620 cells were seeded in each well in 6-well plates and treated with GEN 100 µM for 48 h. Cells were harvested in PBS  with a scraper, and centrifugated at 600 g for 5 min. The pellet was dissolved in RIPA buffer  with protease inhibitors  in a proportion of 100:1. Then, cells were sonicated with Vibra Cell Ultrasonic Processor 75185 on ice in three cycles of 25W for 10 s with an interval of 5 s between each pulse and 40% of amplitude. After that, cells were centrifugated at 600 g for 5 min. The supernatant was recovered and the protein amount was quantified by the BCA method (Thermo Scientific™ 23227), following the manufacturer’s protocol.Total of 8 × 10For all SDS-PAGE carried out, 20 µg of total protein were loaded in each well. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. Proteins were separated by electrophoresis on 12% acrylamide/bisacrylamide (30/1) gel, after that, proteins were electrotransferred, by semi-dry electrotransfer, on a 0.2 µm nitrocellulose membrane  using the Trans-blot Turbo transfer system . Then, membranes were blocked with 5% non-fat powdered milk in TBS-Tween  for 1 h at room temperature and agitation. After that, membranes were incubated over night at 4 °C in agitation with primary antibody . The primary antibodies used and their respective dilutions were: copper–zinc superoxide dismutase (CuZnSOD) 1:1000 , manganese superoxide dismutase (MnSOD) 1:500 , gluthatione peroxidase (GPx) 1:500 , catalase 1:1000 , and GAPDH 1:1000 . Finally, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (2% non-fat powdered milk in TBS-Tween) for 1 h at room temperature and agitation. The secondary antibodies used, and their respective dilutions, were: anti-rabbit 1:10,000 , anti-mouse 1:10,000 and 1:2000 for GPx , and anti-sheep 1:10,000 . To detect the immunoreactivity, Inmun-Star© Western Chemilumeniscence kit Western blotting detection systems  were used. Chemiluminescent signal was acquired with Chemidoc XRS densitometer , and the results were analyzed with Quantity One Software .5 HT29 cells and 8 × 105 SW620 cells were seeded in each well in 6-well plates and treated with GEN 100 µM for 48 h. After that, cells were harvested with trypsin-EDTA and fixed in cold 100% methanol. Fixed cells were incubated at −20 °C overnight and centrifugated for 5 min at 600× g. Before the analysis for DNA staining, cells were incubated at room temperature in the dark for 30 min with an RNAase and propidium iodide mix. Flow cytometry experiments were performed using a Beckton-Dickinson FACSVerse flow cytometer, and the results were analyzed with FACSuite v1.0.6 software.Cell cycle analysis was done by flow cytometry. Total of 9 × 105 HT29 cells and 5 × 105 SW620 cells were seeded on a glass coverslip inside 6-well plates and treated with GEN 100 µM for 48 h. Cells were washed with PBS-T (0.1% Tween 20) and then were fixed with 4% paraformaldehyde  in PBS (pH 7.4) for 10 min at room temperature. After that, cells were washed with cold PBS. Permeabilization was carried out with 0.25% Triton X-100  in PBS for 10 min at room temperature, and then cells were washed with PBS. Then, cells were blocked with 1% BSA  with 22.52 mg/mL glycine  in PBS-T for 30 min at room temperature. After blocking, cells were incubated with NF-κB primary antibody 1:50  with 1% BSA in PBS-T during 1 h in a humidified chamber at room temperature. After incubation, cells were washed with PBS and incubated with anti-rabbit secondary antibody 1:100  with 1% BSA in PBS-T for 1 h in a humidified chamber at room temperature in the dark. Before the DNA staining, cells were washed with PBS. For the DNA staining, cells were incubated with 1 μg/mL Hoechst 33342  in PBS for 1 min at room temperature in the dark and then washed with PBS. Finally, coverslip was mounted with a drop of DAKO Fluorescent Mounting Medium  and incubated O/N at room temperature in the dark.Total of 5 × 10The fluorescence was monitored with a Leica TCS-SPE Confocal Microscope, using 63× immersion oil (147 N.A.) objective lens. Fluorescence emission was 555 nm.5 HT29 cells and 5 × 105 SW620 cells were seeded on a glass coverslip inside 6-well plates and treated with GEN 100 µM for 48 h. Cells were washed with PBS and then were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 10 min at room temperature. After that, cells were washed with PBS and were stained with 0.1 mg/mL Phalloidin–Tetramethylrhodamine B isothiocyanate  for 1 h at 37 °C. After staining, cells were washed with PBS and, for the DNA staining, cells were incubated with 10 mg/mL DAPI  for 10 min at room temperature, and then washed with PBS. Finally, coverslip was mounted with a drop of DAKO Fluorescent Mounting Medium and incubated O/N at room temperature in the dark.Total of 5 × 10The fluorescence was monitored with a Leica TCS-SPE Confocal Microscope, using 63× immersion oil (147 N.A.) objective lens. Fluorescence emission wavelength was 570 nm and fluorescence excitation wavelength was 532 nm.p < 0.05. All results are presented as mean values (n = 6) ± standard error of the mean (SEM).Statistical Program for the Social Sciences software for Windows  was used to perform all the statistical analyses. Student’s t-test was used to analyze the differences between control and treated cells, with minimal statistical significance at 2/M cell cycle arrest and a decrease in cell viability. Although further studies are necessary to better understand the role of genistein during anticancer therapy, this work provides new insights into the effects of high doses of this phytoestrogen on the physiology of colon cancer cells.High concentrations of genistein induced a decrease in cell viability in colon cancer cells that could be promoted by changes in cell cycle regulation. In colon primary cancer HT29 cells, the increase in cell death promotes a slight raise of oxidative stress that can be mitigated by the increase in mitochondrial biogenesis; the increase in ROS production increments the inflammatory status, and finally, results in a moderate decrease in cell viability. On the other hand, in colon metastatic cancer SW620 cells, the significant increase in ROS production, that cannot be palliated by antioxidant enzymes nor mitochondrial biogenesis, produces a great rise in inflammatory levels, that all together results in a G


UCU, CCUG, CAG, and CUG repeats cause spinocerebellar ataxia type 10, myotonic dystrophy type 2, Huntington’s disease, and myotonic dystrophy type 1, respectively. By performing extensive molecular dynamic simulations, we investigated the bending propensities and conformational landscapes adopted by 3×3, 2×2, and 1×1 internal loops observed in RNA AUUCU, CCUG, CAG, and CUG repeat expansions using model systems having biologically relevant repeat sizes. We show that the conformational variability experienced by these loops is more complex than previous reports where a variety of unconventional hydrogen bonds are formed. At the global scale, strong bending propensity was observed in r(AUUCU)10, r(CCUG)15, r(CAG)20, and r(CUG)20, and, to a lesser extent, in r(AUUCU)4, r(CCUG)10, r(CAG)10, and r(CUG)10. Furthermore, RNA CAG repeats exhibit a tendency toward bent states with more than 50% of observed conformations having bending angles greater than 50°, while RNA CUG repeats display relatively linear-like conformations with extremely bent conformations accounting for less than 25% of the observed structures. Conformations experienced by RNA AUUCU repeats are a combination of strongly bent and kinked structures. The bent states in RNA CCUG repeats mostly fall into the moderately bent category with a marginal ensemble experiencing extreme bending. The general pattern observed in all the bent structures indicates the collapse of the major groove width as the mechanical trigger for bending, which is caused by alteration of base pair step parameters at multiple locations along the RNA due to local distortions at the loop sites. Overextension is also observed in all the RNA repeats that is attributed to widening of the major groove width as well as undertwisting phenomenon. This information and the rich structural repository could be applied for structure based small molecule design targeting disease-causing RNAs. The bending propensities of these constructs, at the global level, could also have implications on how expanded RNA repeats interact with proteins.Expansions of RNA AU UCU repeats, r(AUUCU)exp, within intron 9 of the ATXN10 pre-mRNA causes SCA10, a disease with no cures available (CUG), r(CUCG)exp, residing within the intron 1 of the zinc finger 9 (ZNF9) precursor mRNA (pre-mRNA) cause DM2. Expansion of RNA CUG, r(CUG)exp, and CA repeats, r(CAG)exp, cause DM1 and HD, respectively. These expanded RNA repeats fold into structures, which can strongly interact with proteins like heterogeneous nuclear ribonucleoprotein K (hnRNP K) exp and r(CAG)exp have repeating 1×1 UU and 1×1 AA internal loops, respectively, r(AUUCU)exp and r(CCUG)exp possess continuous 3×3 UCU/UCU and 2×2 CU/UC internal loops, respectively, with non-canonical UU, CC, and UC base pairs. As a result, they are highly dynamic and impart far more instability compared with non-canonical 1×1 internal loops.Expanded RNA repeats are responsible for a wide range of neuromuscular diseases such as spinocerebellar ataxia type 10 (SCA10) , myotonivailable . ExpandehnRNP K)  in SCA10hnRNP K) . hnRNP Kpomyosin  and its Inhibition of RNA-protein complexes causing neuromuscular diseases, and hence, restoring cell regulation is a new therapeutic strategy, which has attracted a lot of attention . Among dUCU) and r(CCUG) repeats create challenges while studying the conformational landscapes experienced by these RNA repeats. In the meantime, however, due to their distinct nature, 3×3 UCU/UCU and 2×2 CU/UC internal loop motifs provide unique druggable targets, which could be exploited for therapeutic use. Despite the recent advancements in studying RNA dynamics using NMR exp, r(CCUG)exp, r(CAG)exp, and r(CUG)exp using biologically relevant RNA models  were applied on the RNA molecule in the first step of minimization with 5,000 steps of steepest-descent algorithm and subsequently followed with the second round of minimization with 5,000 steps of conjugate-gradient algorithm and no restraints. Minimization was followed by an equilibration protocol first in constant volume with restraints on the RNA molecule  and gradually increasing the temperature up to 300 K for several nanoseconds using the Langevin thermostat. The second round of equilibration was performed at constant pressure with constant temperature at 300 K and pressure coupling  with a 2 fs time step was performed for each system with isotropic positional scaling. The reference pressure was set to 1 atm with a pressure relaxation time of 2 fs. SHAKE  adopting unique values varying between 10 Å and 18 Å depending on the sequence and the type of the internal loop (see details below).Base pair step parameters, groove widths as well as bending angles, and curvilinear helical axis, were measured using Curves+  and 3DNA+ . The fir+ . Thus, wUCU, and performed 2.9 µs MD simulation to investigate the global structural behavior of AUUCU repeats responsible for SCA10. The 10×AUUCU includes 10 copies of 3×3 UCU/UCU internal loop motifs, which makes it a realistic system to investigate AUUCU repeats. MD trajectory of 10×AUUCU exhibits structures having both bent exp. We performed 2.6-µs-long MD simulation on this system to determine the bending properties of r(CUG)exp. To identify the bending, cluster analyses were conducted on the MD trajectory, average structures for each cluster were calculated, and structural analyses were performed on the average structures using the Curves+. Visual inspection of average structures, which correspond to states with different bending angles  base-paired RNA helixes for 20×CUCU repeats, three RNA constructs each having two, four, and ten copies of AUUCU loops were utilized   . Populatectively . Moreoveectively . It was  minimum  but with minimum . In 4×AUbending. . ResultsCUG repeats, where three models with 4, 10, and 15 copies of CCUG loops were investigated   . Similar limited . RNA CCUnd state . Anotherectively . The casectively . CytidinUG by 8% . Furtherted form . For exase pairs  start toAG and CUG repeat expansions. Again, we used three different model systems in each case having 2, 10, and 20 copies of CAG  and CUG loops   . Previoud states . We obseectively . In one-ectively . Furtherectively , while 0ectively . Similarectively , one of ectively . Analogo systems . This isd states , and samd states .UCU, CCUG, CAG, and CUG repeats have tendencies to bend dramatically with the increase in repeat size. The native form of RNA is A-form, which is a linear structure stabilized by hydrogen bonds formed by Watson-Crick GC, AU, and GU base pairs. Defects in RNA structure such as formation of internal loops can distort the global RNA structure and form states, which are bent or display kink-like structures. RNA repeat expansions have regularly spaced internal loops such as 3×3 UCU/UCU in AUUCU repeats, 2×2 CU/UC in CCUG repeats, 1×1 A/A in CAG repeats, and 1×1 U/U in CUG repeats, which can have global structural implications due to the dynamic behavior of the internal loops exp, r(CUCG)exp, r(CAG)exp, and r(CUG)exp have repeating units with unique internal loops. At atomic level, electrostatics, stacking, and hydrogen bonding interactions are directing the structural preferences of bases in each internal loop, which will create local distortions. The combined effect of all these distortions generates the bending phenomenon discussed above. We already discovered that collapse of Mgw is coordinated with bending phenomenon. Thus, we investigated the connection of Mgw with minor groove width (mgw) and base pair step parameters    in ordernd rise) . One thiUCU, 4×AUUCU, and 10×AUUCU  in 4×AUUCU, and around   and  in 10×AUUCU profiles, where Mgw and θ stand for major groove width and angular change of base pair step orientation , respect10×AUUCU . Results 10×AUUCU. No genu 10×AUUCU.CUG and 15×CCUG display slight differences , , and  profiles calculated for 10×Cferences . For exa 10×CCUG .AG and CUG models are particularly interesting because the 1×1 AA and 1×1 UU pairs in each RNA construct are well defined, which allows base pair step data to be more physical compared with AUUCU and CCUG. For example, analyses display that angular change as well as twist and slide are all important in analyzing properties of RNA CAG repeats    , and   profiles  , and  in 10×CUG and 20×CUG compared with 2×CUG    , and  (UCU)exp, r(CUCG)exp, r(CAG)exp, and r(CUG)exp.The base pair step data of RNA C repeats . Compare −2.5 Å) . Increasth 2×CUG . As befoe = 3 Å) . ResultsUCU, CCUG, CAG, and CUG repeat expansions have repetitive 3×3 UCU/UCU, 2×2 CU/UC, 1×1 A/A, and 1×1 U/U internal loops, respectively, which are dynamic. We found that these RNA repeats have strong bending propensities due to the local structural changes observed in their repetitive internal loop motifs, which collapse the major groove width at different locations in RNA causing global structural change called bending phenomenon. Furthermore, we discovered that there is a link between the magnitude of bending and the system size as well as the RNA internal loop motifs in these RNA repeat expansions, where extreme bending is observed in sequentially long RNA repeats.Bending in nucleic acids, especially in DNA having A-tracts , is a weCUG and AUUCU repeats are alike, we found that latter system exhibited extreme bending regimes as well as kinked conformations. Furthermore, studies on RNA CAG revealed the role of bulky 1×1 A/A mismatches in creating extreme curvatures in RNA structure. We also studied the effect of RNA length on its global behavior in these repeat expansions. We found that extreme bending regimes are only observed in long systems, although bending was also observed in small systems. Analysis of structural parameters revealed that changes in major groove width were the general outcome for observed bent states in all the studied systems. Changes in major groove width are due to weak pairings observed in RNA loop motifs, which can either distort the closing base pairs or the loop structures to provide enough energetics to create local distortions in multiple locations along the RNA resulting bent geometries. Finally, all the model RNA repeats displayed overextension, which is attributed to widening of the major groove widths as well as undertwisting phenomenon. The combined results can provide an understanding on how proteins would interact with RNA repeat expansions. Furthermore, results might be utilized in structure-based drug designs specifically targeting RNA repeat expansions.In summary, we utilized model RNA systems having biologically relevant repeat sizes to provide a glimpse on the behavior of RNA repeats both at global and local levels. Presence of multiple pyrimidine–pyrimidine mismatches in internal loops was found to impose structural stress in RNA resulting in bent or kinked conformations. While RNA C


Notably, LDL treatment increased mRNA levels and insulin secretion; this hyperinsulinism condition was associated with the transcription factor PDX-1. However, a compensatory response that maintains basal levels of intracellular calcium was described, mediated by the overexpression of calcium targets PMCA1/4, SERCA2, and NCX1, together with the upregulation of the unfolded protein response (UPR) through the activation of IRE1 and PERK arms to maintain protein homeostasis. The LDL treatment induced metainflammation by IL-6, NF-κB, and COX-2 overexpression. Furthermore, LDL endocytosis triggered an imbalance of the RAS components. LDL treatment increased the intracellular levels of cholesterol on lipid droplets; the adaptive β-cell response was portrayed by the overexpression of cholesterol transporters ABCA1 and ABCG1. Therefore, lipotoxicity and hyperinsulinism induced by LDL were regulated by the natural compound auraptene, a geranyloxyn coumarin modulator of cholesterol-esterification by ACAT1 enzyme inhibition. EVs isolated from β-cells impaired insulin signaling via mTOR/p70S6Kα in hepatocytes, a phenomenon regulated by auraptene. Our results show that LDL overload plays a novel role in hyperinsulinism, mechanisms associated with a dysregulation of intracellular cholesterol, lipotoxicity, and the adaptive UPR, which may be regulated by coumarin-auraptene; these conditions explain the affectations that occur during the initial stages of insulin resistance.Dyslipidemia is described as a hallmark of metabolic syndrome, promoting a stage of metabolic inflammation (metainflammation) that could lead to misbalances in energetic metabolism, contributing to insulin resistance, and modifying intracellular cholesterol pathways and the renin–angiotensin system (RAS) in pancreatic islets. Low-density lipoprotein (LDL) hypercholesterolemia could disrupt the tissue communication between Langerhans β-cells and hepatocytes, wherein extracellular vesicles (EVs) are secreted by β-cells, and exposition to LDL can impair these phenomena. β-cells activate compensatory mechanisms to maintain insulin and metabolic homeostasis; therefore, the work aimed to characterize the impact of LDL on β-cell cholesterol metabolism and the implication on insulin secretion, connected with the regulation of cellular communication mediated by EVs on hepatocytes. Our results suggest that β-cells can endocytose LDL, promoting an increase in  The relationship among obesity, high levels of Low-Density Lipoproteins (LDL), low levels of High-Density Lipoprotein (HDL), insulin resistance, and arterial hypertension involve a high lipid diet and adipose tissue mass gain, leading to the development of low-grade systemic inflammation. These conditions are associated with the synthesis and release of pro-inflammatory cytokines TNF-α, IL-6, and IL-1 ,3.During the early stage of type 2 diabetes mellitus (T2DM), the synthesis and secretion of insulin by islet β-cells are enhanced, coupled with endoplasmic reticulum (ER) stress , and alt2+ regulates protein synthesis and folding .,30.29,30g for 2 min. The pellet was dissolved in 200 µL of a buffer of 60% sucrose, 10 mM HEPES, 1 mM EDTA, and pH 7.4. After mixing, samples were ice-incubated for 10 min. Next, 800 µL of ice-cold lysis buffer was added and incubated on ice for 10 min. Cells were lysed by 5 passes through a 27-G needle and centrifuged at 100× g for 2 min. A mixture of 2 µL of methylene-blue per mL of lysis buffer was prepared; then, 600 µL of this mixture was carefully layered on top of the cell homogenate and centrifuged at 20,000× g at 4 °C for 120 min. Tubes were frozen at −70 °C and the dye layer containing lipids was recovered (lipid droplets). Triglyceride and cholesterol content of this fraction were measured .Cells were processed as reported elsewhere . Cells wCells were proliferated in 20-mm cell culture plates at a density of 2 × 105 cells/mL, which were processed in a counting-chamber device. Cells were maintained in proliferation for 72 h to reach 90% of confluence, and specific treatments were performed on a volume of 1000 µL. Then, the cell culture medium was recovered and centrifuged for 5 min at 5000 rpm. The supernatant was recovered and diluted in PBS (1/10). Insulin concentrations were quantified with the Ultrasensitive Insulin ELISA kit employing several adaptations according to manufacturer recommendations . Absorbance readings were performed at 450 nm and results were reported as ng/mL.−∆∆Ct method with GAPDH as reference calibrator and reported as fold change. In addition, insulin detection was performed in hepatocytes to validate quantification; the detection was negative.Total RNA from RIN-m5F cells was obtained with Trizol reagent, following the supplier’s instructions. cDNA was synthesized using 1 µg of RNA and the Primer Script RT-PCR Kit . cDNA concentration was standardized for qPCR with the PowerUp Sybr Green Master Mix 2X  according to the manufacturer’s instructions. Primer sequences were insulin forward 5′AGGACCCACAAGTGGAACAACT3′, insulin reverse 5′CAACGCCAAGGTCTTGAAGGT3′, GAPDH forward 5′AGACAGCCGCATCTTCTTGT3′, and GADPH reverse 5′CTTGCCGTGGGTAGAGTCAT3′. qPCR reactions were performed in triplicate using an ABI PRISM 7000 sequence detection system; data were analyzed with the 22+ indicator) in the opti-MEM medium for 75 min. Subsequently, cells were washed with PBS and incubated for 20 min at 25 °C. Cells were washed with PBS and fluorescence measurements were carried out at 340 and 380 nm excitation wavelengths, and a 510 nm emission wavelength, employing a Cary Eclipse fluorescence spectrophotometer . Rmax was obtained with the addition of Triton X-100 (0.035%) and Rmin with the EGTA (4.5 mM) incubation [Cells were processed as previously described , and calcubation . In addiAuraptene (98% purity) was prepared in a 1 mM DMSO stock solution, then evaluated in a concentration range (1–16 µM) on β-cell cultures and concomitant LDL treatment (20 µg/mL). Based on the results of ABCA1 expression and the impact on cholesterol metabolism, we selected a dose of 4 µM to evaluate the protective effect on several indicators such as insulin secretion, intracellular calcium, and EV secretion. In the experiments, DMSO corresponding with a dose of auraptene 4 µM was evaluated as a control.g for 15 min using an S50-A 2559 rotor; subsequently, the pellet was discarded, and the supernatant was recovered. In the next step, the supernatant was centrifuged at 600× g for 30 min, the pelleted cell debris was discarded, then the supernatant was centrifuged at 2000× g for 30 min, later at 10,000× g for another 30 min, and finally at 125,000× g for 80 min in a Sorvall MTX 150 ultracentrifuge. This ultracentrifugation step generates EVs’ pellet [After treatments, β-cells conditioned medium was diluted in PBS and centrifuged twice at 200× ’ pellet . The pelC9 cells were grown on 20-mm well plates  in DMEM supplemented medium. At 90% confluence, C9 cells were incubated under different treatments with 40 μL of EVs isolated from β-cells previously incubated under several conditions. Afterward, the cells were washed with ice-cold PBS and lysed for 35 min at 4 °C with protein-lysis buffer and centrifuged for 10 min at 8000 rpm. The supernatant was recovered, and protein was quantified using a BCA assay. Samples (25 µg) were electrophoresed on SDS-PAGE (10%) gels and transferred to PVDF membranes to evaluate the protein targets of insulin signaling mTOR/p70S6Kα, as well as cholesterol targets.Cell viability was assessed by MTT reduction assay under different conditions for 20 h. First, cells were seeded onto 96-well plates at 23,000 cells/well density and allowed to grow at 90% of confluence. Next, the culture medium was replaced with an Opti-MEM medium. After 2 h under this condition, cells were treated under several experiments. Later, 30 μL of an MTT 2.1 mg/mL stock solution was added to the culture media to obtain a final concentration of 0.5 mg/mL. Formazan crystals formed after 4 h of incubation were further dissolved by adding buffer lysis , according to the previous report . Finallyg. The supernatant was recovered, and the pellet was homogenized in 0.25 mL of SMT buffer by passing them three times through a 21-gauge needle and a 27-gauge needle, centrifuged for 5 min at 1000× g; the resulting supernatant was pooled with the first one. Pellet corresponds to the nucleus, and the supernatant was centrifuged at 25,000× g for 20 min to separate organelles from the cytosol. The pellet was resuspended in 150 µL SMT, loaded on a discontinuous Nycodenz gradient composed of three layers , and centrifuged at 107,000× g for 75 min. The Nycodenz-enriched fraction was recovered and loaded on a 27% Percoll solution, later centrifuged at 35,000× g for 45 min.Cells grown in 100 mm dishes were treated with LDL (25 µg/mL) for 20 h. Later, cell cultures were washed once with cold PBS and scrapped. Cells were homogenized in 1 mL of buffer 0.27 M sucrose and 10 mM MOPS-Tris (pH 6.8) (SMT buffer), passing the cell suspension three times through a 21-gauge needle and a 27-gauge needle. Cell debris and nuclei were removed by centrifugation for 5 min at 1000× Atomic coordinates of protein ACAT/SOAT (PDB ID: 6L47) with a resolution of 3.50 Å were evaluated. The structure of auraptene (CID: 1550607) was obtained from the PubChem database . For docp < 0.01, unless otherwise specified. The software used was GraphPad Prism version 6 . For the analysis of the expression of proteins by Western blot, a semiquantitative analysis was performed using loading controls with the ImageJ software .Data are expressed as mean ± SD. Statistical analyses were conducted with one-way ANOVA, and differences among means were compared with the Bonferroni assay using a significance level of We focused on the effect of LDL treatment on the cholesterol management capability of β-cells. In the first instance, LDL isolation was performed by the KBr ultracentrifugation method and validated through the evaluation of protein targets apoA-1 and apoB-100 on the VLDL, LDL, and HDL fractions. Our results verified the appropriate isolation of the LDL fraction . Accordi2+ has been proposed; for instance, a critical condition of the basal sensitivity of the sterol sensing SREBP activity is the concentration of ER Ca2+ [To gain insights into the mechanisms involved in cholesterol regulation under LDL treatment, we characterized the expression of HMGCR and SREBP2, critical elements for the de novo cholesterol pathway. The data suggest a specific regulation of these targets in β-cells and hepatocytes, wherein cholesterol synthesis could be triggered in β-cells, considering an increased SREBP2 and HMCGR expression C,D. This ER Ca2+ .2+ concentrations. Contrary to expectations, the results showed increased insulin exocytosis activity under the LDL incubation in a concentration-dependent manner (0–40 µg/mL), evaluated by the measurement of insulin concentrations in the extracellular media  was evaluated. PDX-1 is the critical transcription factor for insulin expression and, with the gene BETA2, is associated with β-cell proliferation . LDL tre+, the classical inducer of membrane depolarization  overexpression in hepatocytes, connected with steatosis, activating PERK/CHOP signaling . In β-ceChronic hyperglycemia and hyperlipidemia associated with T2DM disrupt ER homeostasis, which could originate the UPR activation . LDL treAn impaired glucose tolerance and insulin resistance are associated with ACE2-knockout ,47, and To elucidate the potential implication of LDL stimulus on RAS, we focused on the ACE2 primary regulator, the transmembrane Serine Protease 2 (TMPRSS2) D. In the1R dependent on the LDL concentration was registered , we isolated the fat droplet fraction of β-cells according to the procedure reported by Harris et al., 2012 . We idenResults suggest that LDL treatment modified the intracellular cholesterol metabolism, coupled with alterations of insulin secretion and signaling. Under metainflammation, the accumulation of bioactive lipids such as diacylglycerols as a response to triglyceride biosynthesis triggers protein kinase C-ε activation to disrupt insulin signaling . Then, mFerula species [We focused on strategies to inhibit cholesterol esterification and thus prevent its accumulation, inducing mechanisms that carry out cholesterol to the extracellular media through the transporter ABCA1. ACAT can be regulated by an Aur molecule , a geran species . Based o species , conside species .in silico experimentation on an ACAT structure (PDB: 6L47), molecular docking results suggest the interaction of Aur with the residues Arg350, Asn351, Tyr523, Arg525, Gln526, His527, Cys528, and Pro529  [528 and C546) that is not modified by the interaction with Aur [In addition, by employing d Pro529 E,F. The nd W420) . The crywith Aur ,60. An iwith Aur .The regulation of the PERK arm of UPR in β-cells could prevent liver steatosis in mice under a high-fat diet  and, posWe evaluated this proposal employing EVs released under LDL treatment. First, β-cells were incubated for 24 h with LDL (20 µg/mL) and concomitant Aur (4 µM); then, treatments were withdrawn and cells were washed and incubated for another 24 h. Therefore, we ruled out the background effect of LDL. Next, the supernatant medium was recovered and the isolation of the EVs fraction was performed. In the first instance, we confirmed a correct EVs isolation process by characterizing the exosome marker flotillin-2 on several extracellular media taken at different experiment stages. The results showed that adequate EVs fractions could be obtained 24 h after LDL treatment A. LikewiThe effect of EVs isolated from β-cells was characterized under the treatment of hepatocytes cultures for 20 h. We found a diminished insulin signaling phenomenon under the EVs incubation in which vesicles were isolated from β-cells treated with LDL; this phenomenon was characterized by a reduction in the activation of p-p70S6Kα, which was dependent on mTOR signaling, without affecting the expression levels of p70S6Kα C–E. p70S2+ at a resting concentration of ∼100 nM [Based on the characterization of insulin phenomena such as exocytosis and mRNA synthesis, β-cells were highly sensitive to LDL. Therefore, compensatory mechanisms mediated by ABCA1 and calcium-regulator proteins were triggered. Calcium management through the overregulation of SERCA2, NCX1, and PMCA1/4 could maintain free Ca ∼100 nM . Enzymes ∼100 nM . Thus, aBased on our results, a cellular response promoted by high LDL concentrations was the activation of UPR; in this regard, under a saturated lipid stimulus, the UPR sensitization of β-cells by IRE1/XBP1s and PERK/CHOP contributed to the upregulation of inflammatory pathways mediated by NF-κB ,68, impa2+ management proteins and UPR maintains an essential contribution to the regulation of insulin exocytosis. Subtle mechanisms to sustain cholesterol homeostasis involve esterification and hydrolysis [Therefore, the simultaneous function of Cadrolysis ; cholestLDL treatment increased insulin gene expression; however, intracellular calcium and counterbalance mechanisms might be triggered to maintain β-cell proteostasis. Hyperinsulinism was associated with ER stress, metainflammation, and a compensatory response of ABCA1 overexpression. On the other hand, the highly proatherogenic oxidized LDL has been described as downregulating ABCA1 expression via the MEK/ERK/LXR pathway, leading to impaired insulin synthesis and GSIS in a β-cell model . Indeed,Membrane cholesterol content due to hypercholesterolemia has been associated with low insulin secretion and glucose intolerance . LikewisAuraptene treatment ameliorated hyperinsulinism coupled with high ABCA1 levels, which maintained cholesterol homeostasis; therefore, the activity of cholesterol transport and the inhibition of cholesterol esterification through ACAT1 could play a significant protective role. Cholesterol should be the regulatory factor that induces the expression of the PDX-1, impacting hyperinsulinemia. In this regard, the insulin promoter has been described as SREBPs target ; specifiLikewise, an intrinsic connection may exist between the cholesterol regulation and carbohydrate metabolism triggered by factors synthesized in β cells. Our results suggest that the EV originated from metabolic damage in β-cells may contribute to insulin resistance in hepatocytes by decreasing mTOR/p70S6Kα activation. We previously demonstrated that exosomes isolated from supernatants of macrophages exposed to lipopolysaccharides caused a metainflammation phenomenon on hepatocytes, promoting a rearrangement in the cholesterol metabolism . Our eviThe implications between RAS and β-cell metainflammation may impact systemic metabolic regulation and other related mechanisms. The role of HNF1s and ACE2 has been proposed in β-cell activity-dependent glucose homeostasis ,47. A deThe regulation of intracellular cholesterol metabolism represents a fundamental target for insulin secretion, and cholesterol must modulate the concentration of the PDX-1 transcription factor. β-cells showed high metabolic activity, wherein a feedback mechanism in cholesterol management could occur, modifying insulin secretion and activating compensatory mechanisms critical in calcium regulation, proteostasis, and cholesterol homeostasis, mechanisms that allow the maintenance of homeostasis and perform adaptive insulin secretory activity. A concomitant response, originated in β-cells and mediated by EVs, could be triggered in a manner involving tissues such as the liver. The results confirm the role of auraptene on the metabolic effect and implication of LDL on β-cell functionality, maintaining cellular homeostasis, and allow the basis for the use of molecules isolated from natural sources or dietary phytochemicals to be established. Therefore, the results emphasize the importance of strategies to reduce plasmatic LDL concentrations.