Farm animals frequently suffer from chronic inflammatory diseases due to certain physiological or pathophysiological conditions such as weaning, the periparturient period and infections. Traditionally, antibiotics were added to animal diets to counteract inflammation and enhance growth. However, this leads to the emergence of antibiotic-resistant bacterial species which causes potential health hazards. Over several decades, omega-3 polyunsaturated fatty acids have been known to exhibit a multitude of beneficial effects in animal health and are regarded as a functional food with therapeutic potential. We accessed the bioactivity of omega-3 polyunsaturated fatty acids as eicosapentaenoic acid and docosahexaenoic acid in pig intestinal epithelium under different stress conditions in an in vitro set-up. Our results demonstrated the proliferative and cytoprotective properties of the two fatty acids, which are fundamental to determining the cellular mechanism for efficient utilization in pig diets.2O2) in Intestinal Porcine Epithelial Cell line-J2 (IPEC-J2). The results showed that 24 h treatment with EPA or DHA exhibited proliferative effects in IPEC-J2 cells at low to moderate concentrations (6.25–50 μM) (p < 0.05). Further, 24 h pretreatment with individual DHA (3.3 µM), EPA (6.7 µM) or as DHA:EPA  combination increased the mitochondrial activity or cell membrane integrity post-LPS (24 h), DSS (24 h) and H2O2 (1 h) challenge (p < 0.05). Additionally, DHA:EPA  combination decreased the apoptotic caspase-3/7 activity around twofold after 24 h LPS and DSS challenge (p < 0.05). Our study confirms the proliferative and cytoprotective properties of EPA and DHA in IPEC-J2 cells. Increased intracellular mitochondrial activity and cell membrane integrity by ω-3 PUFAs can play a role in preventing enterocyte apoptosis during acute or chronic inflammatory and oxidative stress.Marine and plant-based omega-3 polyunsaturated fatty acids (ω-3 PUFAs) are widely added to animal diets to promote growth and immunity. We tested the hypothesis that eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and their 1:2 combination could counteract acute or long-term damage of lipopolysaccharides (LPS), dextran sodium sulphate (DSS) and hydrogen peroxide (H The IEL maintains intestinal homeostasis by establishing communication between gut microbiota and underlying immune cells. Further, it acts as a “physical barrier” that blocks the entry of luminal pathogens and antigens into the circulatory system . Sometim6 cells per T-75 cm2 culture flask  and maintained at 37 °C under a humidified atmosphere with 5% CO2. At complete confluency, cells were trypsinated with 5 mL trypsin-EDTA . Subsequently, the viable cells were counted in a hemocytometer by tryphan blue  staining and seeded at a density of 3 × 104 cells/well in 96-well plates . Cells were allowed to adhere 24 h prior to the treatments.IPEC-J2 is a non-transformed cell line, derived from the jejunum epithelium of unsuckled piglet . Cell passages of 24–28 were used for the experiments. Cells were cultured in a complete medium consisting of a 1:1 mixture of Dulbecco’s modified Eagle’s medium with stable L-Glutamate and Ham’s F-12 mixture (DMEM/F12) , supplemented with 15 mM HEPES , 5% heat-inactivated fetal bovine serum (FBS)  and 1% penicillin (100 U/mL)/streptomycin (100 mg/mL) . Cells were seeded at a density of 2 × 10Salmonella typhimurium LPS (0–100 µg/mL), DSS (0–10%) for 24 h and H2O2 (0–10 mM) for 1 h. All chemicals were purchased from Sigma-Aldrich .Cells were treated with increasing concentrations of EPA or DHA (0–200 µM) in DMEM medium for 24 h. Similarly, the cells were exposed to different stressors such as 2O2 (1 mM) for 1 h individually to induce inflammatory and oxidative damage.Cells were pre-treated for 24 h with or without DHA (3.3 µM), EPA (6.7 µM) and DHA:EPA  in a DMEM medium, supplemented with 0.05% FBS. The FBS concentration was chosen based on a preliminary study with a range of 0–1% up to 48 h. The concentration of 0.05% FBS optimally supported cell attachment and growth. Subsequently, the cells were challenged with LPS (50 µg/mL), DSS (2%) for 24 h and HCell viability was determined by quantification of mitochondrial oxidoreductase using the -2,5-diphenyltetrazolium bromide (MTT) assay method. Following cellular treatments (1.2b), the supernatant was replaced with 150 µL of MTT buffer  prepared in 1× PBS (0.25 mg/mL) for 2 h. The end product so obtained was dissolved in an equal volume of dimethyl sulfoxide (DMSO) (Sigma-Aldrich), and the optical density (OD) was measured at 570 nm in a calorimetric plate reader . Cells without any treatment were included as a control which represented 100% viability and DMSO was used as blank. Cell viability was calculated using the formula:®, Promega, Madison, WI, USA) in a 96-well plate and incubated for 30 min in darkness, at room temperature. Subsequently, colorimetric measurement was performed in a microplate reader (Bio-Rad) at 490 nm.Cell membrane integrity was estimated by assessing the cytosolic lactate dehydrogenase (LDH) released in the culture media. After the treatments (section 1.2b), 50 µL of the supernatant was mixed with an equal volume of LDH buffer (CytoTox 96The amount of cellular nitric oxide (NO) in the culture media was estimated via the Griess test. For this, 50 µL of supernatant from the treatments (section 1.2b) was incubated with Griess reagent (Promega). The end-point was colorimetrically measured at 490 nm as per the manufacturer’s instructions.4 cells/well in a 96-well plate and the treatments were performed as described above (section 1.2b). Thereafter, supernatant was replaced with assay reagent, incubated in darkness and fluorescence was measured at 499Ex/521Em in a microplate reader  as per manufacturer’s instructions .Cellular apoptosis was determined by accessing the intracellular caspase-3/7 activity. For this, the cells were seeded at a low density of 2 × 1050) were calculated by non-linear regression analysis using GraphPad Prism software (Version 8). Treatment effects were assessed by one-way ANOVA with Bonferroni post-hoc test. At least three independent experiments were performed with three technical replicates. All data are expressed as mean ± SEM and a value of p < 0.05 was considered statistically significant.The values of half inhibitory concentrations (ICp < 0.05). The dose–response curves of LPS, DSS and H2O2 are reported in 2O2 as shown in 50 values (section 1.7) that correspond to 62.02 μg/mL for LPS, 2.07% for DSS and 1.53 mM H2O2, respectively. Based on these values, the concentrations of 50 µg/mL LPS, 2% DSS and 1 mM H2O2 below the IC50 were chosen for further nutrient assessment.As shown in p < 0.05) (2O2 stress (p < 0.05).MTT-based viability assay was used to determine the treatment effects on mitochondrial metabolic activity of IPEC-J2 cells. Pre-treatment of cells with EPA (6.7 µM) and DHA:EPA  increased mitochondrial activity against LPS stress, while DHA did not have an impact compared to the control  a. Simila < 0.05) b and 1 h2 stress c comparep < 0.05). As shown in 2O2 challenge compared to the control . The NO activity in the culture media was tested by Griess reagent. Surprisingly, the NO content in the culture media was below the detection limit (<2.5 µM) for all the treatments (not shown).LDH assay was performed to determine the treatment effects on cell membrane integrity. Neither the individual DHA (3.3 µM)/EPA (6.7 µM) nor its combination DHA:EPA  were able to recover membrane integrity from 24 h LPS a and DSSp < 0.05). Additionally, the reduction of caspase-3/7 activity was comparatively higher for 50 µg/mL over 100 µg/mL of LPS. In contrast, no caspase-3/7 activity was observed for H2O2 challenge (not shown).The intracellular caspase-3/7 activity was estimated to elucidate the treatment effects on apoptosis of IPEC-J2 cells. A twenty-four hours challenge of 50–100 µg/mL LPS a and 2% Supplementation with ω-3 PUFAs of human and animal diets has been reported to control a wide range of inflammatory diseases ,19,20,21Here, we demonstrated the cytoprotective properties of ω-3 PUFAs  in the porcine IPEC-J2 enterocyte model under different biological and chemical stress. IPEC-J2 is a non-cancer and non-transformed enterocyte model. Moreover, it is isolated from the jejunum epithelium, the primary site of nutrients absorption. Furthermore, enterocytes are the major cell population of IEL (~70–80%), making it an appropriate in vitro cell model to delineate nutrient behavior closer to an in vivo environment.2O2 and DSS. LPS is a component of the Gram-negative bacterial cell wall which instigates pro-inflammatory responses by activation of the NF-κB signalling pathway [2O2 is a commonly used chemical in IPEC-J2 cells for the characterization of dietary antioxidants [The pathophysiological events of inflammation and oxidative stress were stimulated in the IPEC-J2 cells by challenge against LPS, H pathway . In addioxidants ,31,32. Ioxidants . DSS is oxidants .50 values were then determined from the viability assay. Subsequently, the stressor concentrations for ω-3 PUFA assessment (section 1.2b) were chosen below the IC50 range.Further, it is important to apply an optimal concentration of the stressor that renders mild toxicity without completely killing or causing irrecoverable cell damage. To estimate this, a dose–response study was performed by exposing the cells to increasing concentrations of each stressor. Their corresponding IC50 range was chosen to investigate the ω-3 PUFA effects against biological damage. Likewise, the concentration of 1 mM H2O2 (1 h) and 2 % DSS (24 h) was chosen to evaluate the ω-3 PUFA effects against chemical damage. Similar concentrations of H2O2 were previously reported to reduce IPEC-J2 viability [We observed that LPS concentrations > 25 µg/mL are required to decrease the viability of IPEC-J2 cells after 24 h exposure. It was reported that high concentrations of LPS between 100 and 300 µg/mL are required to reduce the viability of non-transformed rat IEC-18 enterocytes after 24 h . LPS coniability ,32,33. Aiability .5/Δ6 desaturase enzymes over normal cells [2O2 (1 h) and DSS (24 h) induced stress as observed from the viability assay. Similarly, EPA (6.7 µM) and DHA:EPA  protected the mitochondrial activity against LPS (24 h) stress while single DHA (3.3 µM) administration did not show any impact. Possibly, its concentration is too low to confer cytoprotection unlike EPA or the combination.From the dose–response study of EPA and DHA, a low to moderate concentration between 6.25 and 50 μM was observed to significantly increase viability, while 200 μM exhibited inhibitory effects in IPEC-J2 cells. A similar increase in cell viability at 6.25–25 μg/mL of EPA and DHA was observed in IPEC-1 cells . Furtheral cells ,43,44. Ial cells  and 12.5al cells . Additioal cells ,47,48. O2O2 stress, while they were unable to do so for LPS and DSS stress. The concentrations of LPS and DSS tested in the current study are speculated to have disrupted the IPEC-J2 cell membrane beyond the recovery potential of different nutrient doses tested. Additionally, post-LPS and DSS challenge, a two-fold remarkable decline in apoptotic caspase-3/7 was observed for cells with DHA:EPA  pre-treatment than for those without. In contrast, no caspase-3/7 activity was observed for H2O2. Perhaps the acute challenge of H2O2 activated necrosis instead of apoptosis, which requires further analysis. Generally, cellular apoptosis is activated by multiple pathways such as DNA fragmentation, certain mitochondrial proteins, or electrolyte imbalance, induced by membrane damage [Moreover, as witnessed from the LDH assay, DHA (3.3 µM), EPA (6.7 µM) and DHA:EPA  potentially recovered membrane integrity from He damage . Thus, te damage . Future 2O2. Additionally, for the first time, low concentrations of ω-3 PUFAs were demonstrated to efficiently control both acute and long-term damage. Further, these in vitro effects should be translated into in vivo efficacy through animal trials for dose–effect optimization.To summarize, our current study demonstrated the proliferative and cytoprotective properties of ω-3 PUFAs as EPA and DHA in the non-transformed porcine enterocyte model, IPEC-J2. EPA and DHA, either individually or in combination, are able to suppress the enterocyte apoptosis through recovery of mitochondrial activity or cell membrane integrity from the detrimental effects of LPS, DSS and H


Hmox1-deficient  pregnancies is impaired, with morphological defects similar to Hif1α-deficient placentas.Severe hypoxia exists in placentas during early pregnancy, with reoxygenation during mid-gestation. Hypoxia-inducible factor-1α (Hif1α), an oxygen sensor, initiates placental vascular development. We have shown that the placental vasculature in 2) and E9.5–15.5 (8%–10% O2). mRNA levels were determined using real-time RT-PCR or PCR arrays and protein levels using Western blot. Bone marrow-derived macrophages (BMDMs) from WT, Het, and Hmox1 knockout (KO) mice, representing different Hmox1 cellular levels, were generated to study the role of Hmox1 on Hif1α ′s response to hypoxia-reoxygenation and gestational age-specific placental lysates.Whole wild-type (WT) and Het mouse placentas were collected at E8.5 , antioxidants , and transcription factors  were significantly different in Het placentas. In response to in vitro hypoxia-reoxygenation and to WT or Het placental lysates, Hif1α transcription was lower in Het and Hmox1 KO BMDMs compared with WT BMDMs.Hif1α was expressed in WT and Het placentas throughout gestation, with protein levels peaking at E8.5 and mRNA levels significantly upregulated from E9.5–E13.5, but significantly lower in Het placentas. Genes associated with angiogenesis  placentas is gestational age-dependent.Expression of •Hif1α expression is reduced in Hmox1-deficient placentas.•Hmox1-deficient placentas.Expression of angiogenic genes is altered in •Hif1α in cells.Hypoxia-reoxygenation induces a differential expression of •Hif1α in Hmox1-deficient cells.Adding placental lysates dysregulates expression of  ARNTaryl hydrocarbon receptor nuclear translocatorBMbone marrowBMDMsbone marrow-derived macrophagesCOcarbon monoxideDFOdeferoxamine mesylate saltHmoxheme oxygenaseHet+/-Hmox1Hifhypoxia-inducible factorHREShypoxic response elementsKOknockout2OoxygenROSreactive oxygen speciesSAspiral arteryuNKuterine natural killerWTwild-type12] or ~18 mmHg) microenvironment is present in the placenta due to the formation of a trophoblast plug within spiral arteries (SAs), which prevents the flow of maternal blood into the fetoplacental interface. During the 2nd trimester, the plug dissolves and maternal blood can now enter the intervillous spaces, thereby increasing O2 levels to 8% (60 mmHg) [2 levels increase (8%), the trophoblast stem cells begin to differentiate into their invasive phenotype, which can migrate and invade into SAs, and stimulate maternal and fetal blood vessel growth [In early pregnancy, a very critical window for placental development exists in both the human and mouse. During the 1st trimester, a severe hypoxic (1–3% oxygen [O60 mmHg) . This no60 mmHg) . When O2l growth .2 tensions is hypoxia-inducible factor (Hif) [2, Hif1α is degraded rapidly in the presence of O2.The primary cellular molecule that senses and responds to Oor (Hif) . Hif is or (Hif) . Therefoor (Hif) ,16. Hif 2 level in the placental microenvironment is 2%–3% and at low levels at 10–12 weeks of gestation when the O2 level is 8% to 10% [Hif1 knockout (KO) mice showed that the disruption of either the Hif1α or Hif1β subunit in fetal trophoblasts results in improper placental development, which is characterized by an insufficient chorion/allantois fusion and a diminished spongiotrophoblast and labyrinth layer [Hif1α expression in either the maternal or fetal side of the placenta may be associated with impaired vascular development similar to that seen in preeclampsia [Hif1α is found at high levels at 7–9 weeks of gestation when the Oth layer ,18. Moreth layer . Therefoclampsia .Hmox1, the inducible isoform, has been associated with a number of human pregnancy disorders [Hmox1-deficient  mice have preeclampsia-like features, such as hypertension and high sFlt 1 levels [Hif1α-deficient mouse.Heme oxygenase (Hmox) is the enzyme that degrades heme to produce equimolar amounts of carbon monoxide (CO), iron, and biliverdin/bilirubin . It is aisorders . In addi1 levels ,28. Het 1 levels ,29,30. H1 levels . InteresHmox1 induces the dysregulation of Hif1α, which subsequently causes placental malformation and dysfunction. To this end, we compared Hif1α protein and mRNA levels throughout gestation between wild-type (WT) and Het placentas and investigated the response of Hif1α to either hypoxia-reoxygenation or placental lysates in bone marrow-derived macrophages (BMDMs) generated from WT, Het, and Hmox1 KO mice.Therefore, we hypothesized that a deficiency in 2All studies were approved by Stanford University's Institutional Animal Care and Use Committee (Protocol #14525) and conducted in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals.2.1Hmox1 Het pairs were used from our mouse colony. The original Hmox1 KO (Hmox1−/−) mouse strain has a targeted deletion of a large portion of Hmox1 gene, and was created on a C57BL/6 background. To establish our Het colony, C57BL/6 Hmox1 mice were backcrossed with FVBn WT mice for >6 generations [Hmox1 KO mice were genotyped using PCR as described previously [Adult WT FVBn mice were purchased from Charles River Laboratories . Adult female and male erations . WT or Heviously .2.2Hmox1, Hmox2, Hif1α, Hif2α, angiogenic factors , receptors ; antioxidant proteins , cyclin-dependent kinase inhibitor 1 , transcription factors , and housekeeping genes (Actb and Gapdh) [To create a customized PCR array, primers were chosen from the PrimerBank-MGH-PGA  and validated by PCR screening. Specific genes were selected that encode for proteins: d Gapdh) . Primer Hmox1 KO placentas  at different gestational ages using a Trizol Reagent . cDNA was synthesized using a RT2 First Strand Kit . Real-time RT-PCR was then performed using the RT2 Real-Time SYBR Green/ROX PCR Master Mix (Qiagen) on a Stratagene Mx3005P QPCR system . Data were then analyzed using the ΔΔCt method, normalized to Actb and Gapdh. Fold change in gene expression over control levels were then calculated.Total RNA was extracted from whole WT, Het, and 2.3Whole placental samples were sonicated and prepared as previously described . 2 mM de2.4Hmox1 KO mice as described [7 BM cells in 10 ml media (106 cells/ml) were seeded onto 100-mm Petri-dish plates  and maintained in 20% L929 conditional RPMI medium with β-mercaptoethanol. At day 3, half of the media was replaced with fresh media. At day 7, attached cells were treated with IL-4  to differentiate M1 cells into M2a cells.BMDMs were generated from bone marrow (BM) cells isolated from WT, Het, and escribed . In brie2.52.5.12 concentration of 21%, 5%, 1%, or 1%–21% using a hypoxia incubator (Thermo Scientific).M2a cells were exposed for 24 h at an O2.5.2Placentas from pregnant WT and Het mice at E8.5 or E10.5 were collected in RPMI medium. Tissues were homogenized, centrifuged, and filtered through 0.22-μm columns to yield crude lysates for treatment of BMDMs.2.6For comparisons of experimental groups, one-way ANOVAs were first performed for each set of experiments to determine significant differences when P < 0.05. To determine differences between individual experimental and control groups, Dunnett's tests were used which adjusts for multiple comparisons using the same control group.33.1Hif1α expression in WT placentas change throughout gestation, we collected placentas at E8.5 to E17.5 and measured Hif1α mRNA levels using real-time RT-PCR. Significant upregulation of Hif1α mRNA was observed beginning at E9.5, when O2 levels are at 8% compared with O2 levels of 1%–3% at E8.5. Levels peaked between E10.5 to E11.5, and gradually decreased thereafter  or induced  compared with WT placentas. The alterations in Vegfa, Vegfr1, and Mmp2 occurred early in pregnancy (E8.5 to E10.5), while changes in Cxcl12, Angpt1, and Nos3 were primarily observed at mid-to-late gestation (E11.5 to E15.5) .Using a custom-made PCR-array , we compo E15.5) A. In addlacentas B. Expreslacentas C. Hmox1 3.4Hmox1 deficiency underlies the differential expression of Hif1α in response to hypoxia-reoxygenation, we first generated BMDMs from non-pregnant WT, Het, and Hmox1 KO mice and then treated these cells with IL-4 to differentiate these M0 cells into M2a cells. These cells were further exposed to different oxygen tensions: 21%, 5%, 1%, and 21% after 1% pretreatment. Our experimental design is outlined in Hmox1 deficiency in Het and Hmox1 KO cells were confirmed by RT-PCR , but less increased in Het cells (~2-fold). However, Hif1α was not induced in Hmox1 KO cells (~0.5 fold), suggesting that the response of Hif1α to hypoxia-reoxygenation is regulated by the level of Hmox1 expression.To further investigate whether y RT-PCR B. When cenotypes C. Compar3.5Hif1α expression in Het placentas was due to insufficient Hmox1 expression in the cells or due to the Hmox1 deficiency-induced alterations in the placental microenvironment, we generated BMDMs from WT, Het, or Hmox1 KO mice and exposed these cells with lysates prepared from WT or Het placentas harvested at two gestational ages, E8.5 and E10.5, when O2 levels are low and high, respectively , growth factors, and inflammatory cytokines. Due to their ability to induce Hif1α expression, these factors are also called “pseudo-hypoxia factors” [st trimester, placental O2 levels rapidly increase, which can then lead to a transient period of oxidative stress as well as increased inflammatory cytokine levels. This is supported by our observations that certain antioxidant genes, such as Sod1 and Hmox1, significantly increase from E9.5 to E11.5 (Hif1α was found in the < 3-kd fraction (unpublished data), which contains the highest levels of ROS.The upregulation of factors” . We specto E11.5 B. In addHIF1α has been shown to regulate Hmox1 expression [Hmox1 levels on Hif1α expression has not as yet been elucidated. Here, we show evidence that Hmox1 deficiency could induce the dysregulation of Hif1α in the placenta. Compared with WT placentas, significantly lower Hif1α mRNA and Hmox1 protein levels were found in Het placentas throughout all gestational ages  compared with WT placentas   A, suggesHif1α expression in Hmox1 deficiency placenta is primarily due to the manipulation of the cellular molecular signaling or the changes of placental microenvironment, we used cultured WT, Het, and Hmox1 KO BMDMs treated with different O2 tensions or lysates from WT or Het placentas. BM and its derived cells (BMDMs) are relatively easier to collect, differentiate, and maintain compared with other primary cells, such as trophoblasts or endothelial cells. In these studies, we found that a deficiency of Hmox1 (either Het or KO) alone is not associated with the differential Hif1α expression at either normoxia or hypoxia; but only at the transition state from hypoxia to reoxygenation  affected by Hmox1 deficiency was not possible. Previous work has shown that Hmox1 is expressed primarily by trophoblast cells, such as invasive trophoblasts and spongiotrophoblast cells [Hif1α is caused by a deficiency in Hmox1, which could also occur in these trophoblast cells, and are well known for their role in SA remodeling, the failure of which is likely a contributing cause of preeclampsia.We are also aware of the limitations of this study. Only whole placental tissues were used for measurements of st cells . Thus, w2 tensions in early pregnancy, followed by an increased in vascular formation via angiogenesis in later pregnancy. Therefore, the dysregulation of Hif1α, resulting in either a down- or upregulation, would have detrimental effects on SA remodeling and subsequent abnormal fetoplacental vascular formation [Hmox1 could alter Hif1α expression in response to O2 tensions in the placental microenvironment and cause an alteration of the downstream genes associated with angiogenesis. Our model has provided an example of how pregnancy disorders can be affected by a dysregulation of Hif1α induced by other genetic mutations. Further research is needed to identify other gene or combinations of genes that can also affect Hif1α expression in response to hypoxia-reoxygenation, improving our understanding of placental pathology and possibly guiding therapeutic approaches to various pregnancy disorders involving placental vascular maldevelopment.The establishment of the fetomaternal interface is intricately regulated by Oormation . Our fin10.13039/100000912March of Dimes Prematurity Research Center at Stanford University; the Charles B. and Ann L. Johnson Research Fund; the Christopher Hess Research Fund; the Providence Foundation Research Fund; the Roberts Foundation Research Fund; the Stanford Maternal and Child Health Research Institute; and the 10.13039/100000865Bill and Melinda Gates Foundation.This work was supported by the Prematurity Research Fund; the All the authors confirm that they have the guidance on competing interests and none of the authors have any competing interests in the manuscript.


Rift Valley fever (RVF) is a zoonotic, viral disease, transmitted by mosquitoes, characterized by high mortality rates in young animals. RVF is an endemic and enzootic disease inthe Arabian Peninsula and Africa, causing public health and economic instability. Therefore, it is important to develop vaccines to minimize outbreaks and combat the disease. Wedocumented the stability of the thermo-stability of live attenuated RVF CL13T and recombinant arMP-12ΔNSm21/384 vaccine candidates at different temperatures, including these vaccineviruses in liquid and lyophilized form. The study revealed that both CL13T and recombinant arMP-12ΔNSm21/384 strains were stable for more than 18 months at 4°C. We show thatat room temperatures (37°C and 45°C) the CL13T was less temperature sensitive than MP-12NSm-del in both lyophilized and liquid form. These findings are useful for the preparationof RVF vaccines that will avoid the need for a cold chain and therefore, will improve the application of the vaccines under field conditions. Rift Valley fever virus (RVFV) is a member of the order Bunyavirales, family Phenuiviridae, genus Phlebovirus ,2,the vVaccination is considered an effective strategy against RVF. Several vaccines have been developed. The available live attenuated Smithburn vaccine that originated from South Africa,have been used in Africa countries and Saudi Arabia, however, this vaccine cause abortions and teratogenic effects . The RVFThe thermo-stable RVFV Clone 13T vaccine (CL13T) was derived by cloning the CL13 virus that was isolated from a human case as a naturally attenuated strain and was rendered thermostableby three cycles of heating (56°C) and selection of thermotolerant particles. The resulting candidate vaccine (CL13T) was stable at 4°C for 20 months and shows significantly improvedlevels of thermostability over the existing the CL13 vaccine when tested after exposure to different temperatures . A reverThe two vaccine viruses were produced separately on Vero cells maintained in Dulbecco's modified minimum essential medium (DMEM) containing 10% fetal bovine serum (FBS). The culturemedium from the Vero cells was removed and replaced with an inoculum of RVFV CL13T or arMP-12ΔNSm21/384 at a multiplicity of infection (MOI) of 0.01. After 1 hour of incubationat 37°C, the inoculum was removed and replaced with 10 mL of DMEM with 10% FBS and incubated at 37°C for 2 to 3 days until cytopathic effect (CPE) became apparent. When cellsshowed CPE, the supernatant was harvested and formulated by mixing V/V the viral suspension of CL13T or arMP-12ΔNSm21/384 with a stabilizer of lyophilization .The two freeze-dried RVFV vaccine were tested for stability at 4°C for 18 months and at an accelerated process by repeated titrations to determine infectivity titers after exposurein water bath temperatures of 24°C, 37°lC and 45°C, with aliquots of virus titrated every 3 days for 15 days and daily for 4 days respectively. After the reconstitution ofthe freeze-dried vaccine viruses in the diluent ,aliquots in the glass vials were prepared and stored in incubators at 24°C, 37°C and in a water bath at 45°Cand then tested by periodic titration to determine infectivity titers:[1] At 4°C and 24°C, with aliquots of virus titrated at 2, 4, 7 and 10 days post exposure.[2] At 37°C, with aliquots of virus titrated at 6, 12, 18, 24 and 32 hours post exposure.[3] At 45°C, with aliquots titrated for 2, 4, 6, 12, 14 and 24 hours post exposure.The thermal stability evaluation of the two vaccines CL13T and arMP-12ΔNSm21/384 was repeated 3 times under the same conditions as used in the first or should it be in theseexperiments. The average of the 3 infectivity titers was calculated at each exposure time.Tissue Culture Infectious Dose 50% Endpoint (TCID50) Titration Method.The TCID50 endpoint infectivity titration method was used in this study . The verThe infectivity titers of the freeze-dried RVFV vaccine viruses were was determined by titration on Vero cells using the TCID50 method. The contents of two vials of the reconstitutedfreeze-dried vaccine viruses were diluted 10-fold diluted until 10-3 and 100 µl of each dilution was added to wells of 96-well plates, 6 wells per dilution. Virus dilution was carriedout on ice. Then, 150 µL of Vero cell suspension were added to each well (120000 cell/mL) and the cells and inoculum were incubated at 37°C, 5% CO2 for 5 to 7 days.The cells were examined for cytopathic effect (CPE), and the cells with a CPE were considered positive and those without CPE were considered negative. The titer of the each vaccinevirus was determined using the Reed-Muench method .The infectivity titer of freeze-dried and liquid form at different temperature of storage was compared between the RVFV CL13T and arMP-12ΔNSm21/384 vaccine viruses using thet-test. A p-value ≤ 0.05 was statistically significant.The freeze-dried RVFV vaccine viruses CL13T and arMP-12ΔNSm21/384 remained stable at 4°C for more than 18 months (data not shown). At room temperature (24°C),the infectivitytiters for both viruses decreased by 0.4 logs after 3 days of storage, then the titer remained stable during 15 days for CL13T and 12 days for MP-12NSm-del. At 37°C, the titer ofthe freeze-dried vaccines decreased from 106.4 to 103.5 TCID50/mL for CL13T and from 106.8 to 104.0 TCID50/mL for the arMP-12ΔNSm21/384. At 45°C, the CL13T showed a decreaseof 2.8 logs TCID50, and the arMP-12ΔNSm21/384 decreased by 3.3 log TCID50 in 4 days .After reconstitution in the diluent at 4°C, the CL13T and arMP-12ΔNSm21/384 were stable for 10 days and, 7 days respectively. At room temperature, the CL13T lost 0.8 login viral titer whereas the arMP-12ΔNSm21/384 lost 2.8 logs in 4 days. At 37°C, the titer of CL13T dropped by 0.4 log (from 105.2 to 104.8) in the first 12 hours and then stabilizedat 104.5 TCID50 for more than 32 hours. For the same duration, the arMP-12ΔNSm21/384 titer decreased from 5.4 to 3.5 logs. At 45°C, the CL13T remained stable for more than 3hours, then the virus titer dropped 2.2 log after 32 hours of exposure, during which time the arMP-12ΔNSm21/384 loss all infectivity .This stNo significant difference was observed between the decrease in infectivity titer of the RVFV CL13T and arMP-12ΔNSm21/384 vaccine viruses at 4°C and room temperature forboth liquid and lyophilized forms and at 37°C for the lyophilized form. However, a significant difference (p<0.05) was found between the decrease in titer of the RVF CL13T andarMP-12ΔNSm21/384 at 45°C (liquid and lyophilized forms) and at 37°C (liquid form). The decrease in virus titer after lyophilization, the storage conditions and/or anincapability to preserve the cold chain during the vaccine delivery are problems facing the use of live vaccines. A formalin-inactivated vaccine, commercially available from the OnderspoortVeterinary Institute in South Africa for veterinary use, was reported to be more stable than the live attenuated form of this vaccine ,StudiesThe currently available CL13 attenuated vaccine has been shown to be effective in sheep, goats and cattle ,but is nOur analysis shows that both the CL13T and recombinant arMP-12ΔNSm21/384 vaccine viruses were stable for more than 18 months at 47deg;C. We also show that at room temperatures(37°C and 45°C) the CL13T was less temperature sensitive than arMP-12ΔNSm21/384 in both lyophilized and liquid form. These findings are useful for the preparation of RVFvaccines that will avoid the need for a cold chain and therefore, will improve the application of the vaccines under field conditions.


Several studies have previously documented the role of glycosylation in the expression and functioning of Cav3.2 and suggested that altered glycosylation of the channel may contribute to the aberrant expression of the channel in diabetic conditions. In this study, we aimed to analyze the expression of glycan-processing genes in DRG neurons from a leptin-deficient genetic mouse model of diabetes (db/db). Transcriptomic analysis revealed that several glycan-processing genes encoding for glycosyltransferases and sialic acid-modifying enzymes were upregulated in diabetic conditions. Functional analysis of these enzymes on recombinant Cav3.2 revealed an unexpected loss-of-function of the channel. Collectively, our data indicate that diabetes is associated with an alteration of the glycosylation machinery in DRG neurons. However, individual action of these enzymes when tested on recombinant Cav3.2 cannot explain the observed upregulation of T-type channels under diabetic conditions.CaAbbreviations: Galnt16: Polypeptide N-acetylgalactosaminyltransferase 16; B3gnt8: UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 8; B4galt1: Beta-1,4-galactosyltransferase 1; St6gal1: Beta-galactoside alpha-2,6-sialyltransferase 1; Neu3: Sialidase-3 This  [ob/ob) . Further in vivo , suggestv3.2 channels [v3.2 channel may undergo aberrant glycosylation during diabetes have remained unknown.Protein glycosylation is a posttranslational modification that refers to the co-valent addition of a sugar molecule oligosaccharide (glycan) to specific residues within the target protein. It is an essential chemical process that contributes to the proper maturation, sorting, and functioning of proteins including ion channels ,7, and schannels ,9. Howevdb/db mice and assess the effect of these enzymes on the expression of recombinant Cav3.2 channels.In this study, we aimed to specifically analyzed the transcriptomic profile of glycan-modifying enzymes in DRG neurons from diabetic db/db mice and their control hibernates were purchased from Janvier Labs and were kept under standard conditions for 3 weeks to allow sufficient adaptation. The mean glycemia values measured using a glucocard X-meter ARKAY from blood samples drawn from the tail were 8.2 ± 0.6 mmol/L for wild-type animals (n = 6) and 29.9 ± 0.7 mmol/L for db/db animals (n = 7).8 weeks old male db/db mice using the Glycosylation RT2 Profiler PCR Array (Qiagen) according to the manufacturer’s instructions. The PCR array and qRT-PCR were performed on a LightCycler® 480 (Roche) with the following PCR conditions: 95°C for 5 min, 40 cycles at 95°C for 15 sec, 60°C for 15 sec, and 72°C for 20 sec. Each test was run three times and the mean values were taken to eradicate any discrepancies. 84 key genes encoding glycan-processing enzymes were analyzed and included glycosyltransferase and glycosidase for several important sugars .Transcriptomic analysis of glycan-modifying enzymes was performed on total RNA harvested from the dorsal root ganglia (lumbar L4/L6) of wild-type and v3.2 wild-type in pcDNA3.1 was previously described [The cDNA construct encoding for the human Caescribed . The plav3.2 plasmid and 2.5 ± g of plasmid encoding for the glycan-modifying enzymes. For transfections using the channel alone, 2.5 ± g of empty pcDNA3 vector was added to the mixture to maintained the total amount of cDNA.Human embryonic kidney tsA-201 cells were grown in high glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin  and maintained under standard conditions. Cells were transfected using the calcium/phosphate method using 2.5 ± g of Cav3.2 channels was performed 72 h after transfection in the whole-cell configuration at room temperature (2224°C) as previously described [2, 5 KCl, 1 MgCl2, 128 NaCl, 10 TEA-Cl, 10 D-glucose, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.2 with NaOH). Patch pipettes were filled with an internal solution containing (in millimolar): 110 CsCl, 3 Mg-ATP, 0.5 Na-GTP, 2.5 MgCl2, 5 D-glucose, 10 EGTA, and 10 HEPES (pH 7.4 with CsOH), and had a resistance of 2–4 MΩ. Recordings were performed using an Axopatch 200B amplifier (Axon Instruments) and acquisition and analysis were performed using pClamp 10 and Clampfit 10 software, respectively (Axon Instruments). The linear leak component of the current was corrected online and current traces were digitized at 10 kHz and filtered at 2 kHz. The voltage dependence of activation of Cav3.2 channels was determined by measuring the peak T-type current amplitude in response to 150 ms depolarizing steps to various potentials applied every 10 s from a holding membrane potential of −100 mV. The current-voltage relationship (I/V) curve was fitted with the following modified Boltzmann Patch clamp recording of T-type currents in tsA-201 cells expressing Caescribed . The extI(V) being the peak current amplitude at the command potential V, Gmax the maximum conductance, Vrev the reversal potential, V0.5 the half-activation potential, and k the slope factor. The voltage dependence of the whole-cell Ba2+ conductance was fitted with the following modified Boltzmann with G(V) being the Ba2+ conductance at the command potential V.with v3.2 channels was ascertained by measuring the peak T-type current amplitude in response to a 150 ms depolarizing step to −20 mV applied after a 5 s-long conditioning prepulse ranging from −120 mV to −30 mV. The current amplitude obtained during each test pulse was normalized to the maximal current amplitude and plotted as a function of the prepulse potential. The voltage dependence of the steady-state inactivation was fitted with the following two-state Boltzmann function (3):The voltage dependence of the steady-state inactivation of CaImax as the maximal peak current amplitude and V0.5 as half-inactivation voltage.with The recovery from inactivation was determined using a double-pulse protocol from a holding potential of −100 mV. The cell membrane was depolarized for 2 s at 0 mV (inactivating prepulse) to ensure complete inactivation of the channel, and then to −20 mV for 150 ms (test pulse) after an increasing time period (interpulse) ranging between 0.1 ms and 2 s at −100 mV. The peak current from the test pulse was plotted as a ratio of the maximum prepulse current versus interpulse interval. The data were fitted with the following single-exponential function (4):where τ denotes the time constant of channel recovery from inactivation.n measurements. Statistical analysis was performed using GraphPad Prism 7. Statistical significance was determined using a one-way ANOVA test and datasets were considered significantly different for p ≤ 0.05.Data values are presented as mean ± S.E.M. for db/db) versus wild-type animals (p < 0.05) in diabetic conditions  responsible for catalyzing the initial addition of N-acetylgalactosamine to a serine or threonine residue on early protein precursors [N-acetylglucosaminyltransferase) responsible for the elongation of the polylactosamine chains on tetraantennary N-glycans [N-glycans in the Golgi apparatus [v3.2 with glycosyltransferases  and sialic acid-modifying enzymes  are shown in v3.2 nearly abolished T-type currents. For instance, the maximal T-type conductance (Gmax) in cells expressing Cav3.2 with Galnt16, B3gnt8, and B4galt1 was reduced by 98% (p = 0.0001) , 92% (p = 0.0003) , and 80% (p = 0.0002) , respectively, compared to cells expressing Cav3.2 alone    (p = 0.7542) of Gmax in cells co-expressing the sialidase Neu3   . We also n = 13) . In cont n = 24) . Altogetv3.2 channels in the presence of the sialyltransferase St6gal1 and sialidase Neu3. The mean half-activation potential in cells expressing St6gal1 was shifted by 4.9 mV (p = 0.0001) toward depolarized potentials  compared to cells expressing the channel alone   . In conttivation  or the rchannels . Altogetv3.2 in primary afferent nociceptive fibers is causally linked to the development of peripheral painful neuropathy associated with nerve injury [v3.2 and alteration of the posttranslational regulation of the channel including ubiquitinylation [v3.2 was proposed to contribute to the sensitization of nociceptive fibers in response to hyperglycemia associated with diabetes [db/db mice compared to wild-type animals. These results are consistent with previous studies reporting an alteration of glycan-processing enzymes in the kidney of diabetic mice [in vivo [v3.2 when co-expressed individually with the channel. For instance, co-expression of glycosyltransferases Galnt16, B3gnt8, and B4galt1 with Cav3.2 produced an almost complete loss of functional expression of the channel. However, several studies have previously shown that glycosyltransferases can form heterodimers that contribute to their subcellular expression, enzymatic activity, efficient biosynthesis of glycan chains, trafficking through intracellular vesicles, and substrate specificities [v3.2 in tsA-201 cells as performed in our study may not fully capture the more complex situation in DRG neurons where the expression several genes encoding for glycan-modifying enzymes is altered at the same time and there could be synergetic effects among the various players. Furthermore, tsA-201 cells were grown in high glucose medium which represents another variable that could have influenced the phenotypic effect of these enzymes on Cav3.2 channels. In contrast to glycosyltransferases, co-expression of the sialyltransferase St6gal1 produced a relatively mild decreased expression of the channel with a depolarized shift of the voltage-dependence of activation, indicating that sialylation contributes to the functioning of Cav3.2. However, co-expression of the neuraminidase Neu3 that removes sialic acid moieties did not altered expression of the channel, nor its gating properties. These results are consistent with previous studies showing that application of neuraminidase on tsA-201 cells expressing Cav3.2 channels did not alter channel function [Increased expression of Cae injury –20, antie injury –23, infle injury ,25, and e injury ,3. Severnylation , SUMOylanylation  and phosnylation ,29. Defetic mice . Several[in vivo . Howeverficities . For insficities . Therefov3.2 channels. At this stage, we cannot exclude that expression of glycan-modifying enzymes in DRG neurons may have produced a different phenotypic effect on Cav3.2 and this aspect would deserve further investigations in native conditions.Altogether, this study identified several glycan-modifying genes whose expression is altered in DRG neurons under diabetic condition. However, we did not find evidence for a role of these enzyme in the up-regulation of Ca


In the case of fire, explosive spalling often occurs in cementitious composites due to dense microstructure and high pore-pressure. Polymer fibers were proved to be effective in mitigating such behavior. However, deterioration of these fiber-reinforced cementitious composites inevitably occurs, which is vital for the prediction of structural performance and prevention of catastrophic disaster. This paper concentrates on the behavior and mechanism of the deterioration of polyvinyl alcohol fiber-reinforced engineered cementitious composite (PVA-ECC) after exposure to elevated temperatures. Surface change, cracking, and spalling behavior of the cubic specimens were observed at room temperature, and after exposure to 200 °C, 400 °C, 600 °C, 800 °C, and 1200 °C. Losses in specimen weight and compressive strength were evaluated. Test results indicated that explosive spalling behavior was effectively prevented with 2.0 vol% polyvinyl alcohol fiber although the strength monotonically decreased with heating temperature. X-ray diffraction curves showed that the calcium hydroxide initially decomposed in the range of 400–600 °C, and finished beyond 600 °C, while calcium silicate hydrate began at around 400 °C and completely decomposed at approximately 800 °C. Micrographs implied a reduction in fiber diameter at 200 °C, exhibiting apparent needle-like channels beyond 400 °C. When the temperature was increased to 600 °C and above, the dents were gradually filled with newly produced substance due to the synergistic effect of thermal expansion, volume expansion of chemical reactions, and pore structure coarsening Concrete, a traditional building material, has been recognized as a quasi-brittle material with low ductility and high brittleness, which is harmful to the serviceability and durability of concrete structures. To overcome these drawbacks, engineered cementitious composites (ECC) with strain-hardening features have been developed over the last three decades ,2,3. It However, wider applications would increase the probability of PVA-ECC encountering the risk of fire or high temperature. When on fire, the maximum indoor temperature of a building structure could reach as high as 800 °C to 1000 °C, the mechanical properties of cement-based materials become seriously damaged, and the bearing capacity and deformability of structure elements will also be greatly weakened. During the last decade, over 310,000 fire disasters occurred in China, i.e., nearly 8.5 cases per day, and more than 1269 people died in home fires in the year of 2016, not to mention the economic loss. Therefore, fire resistance and the degradation mechanism of cementitious materials are of vital importance to structural safety and have attracted ever-increasing attention recently ,22,23. CFor concrete, the residual compressive strength after experiencing 400 °C is generally reported around 80%. However, the reduction rate of strength beyond this temperature increases dramatically, resulting in less than 10% of residual strength compared to that at ambient conditions in the range of 800–900 °C . Figure Nonlinear behavior was observed for all curves and the peak stress and stiffness decreased with the increase in heating temperature (except for 200 °C), whilst the corresponding strain increased. Such phenomena were also reported by other authors ,27. It i2O3∙3CaSO4∙32H2O), calcium hydroxide (CH), and calcium silicate hydrate (C-S-H) gels were reported to be initiated at 70, 430, and 600 °C, respectively [The behaviors of PVA-ECC under fire/high temperatures are believed to be related to microstructure changes in cementitious composites at elevated temperatures. For instance, the decompositions of ettringite  and microstructure change need to be established. To achieve this, changes in mechanical performance and microstructure of PVA-ECC subjected to different elevated temperatures were focused on in the present paper. Color changes and surface cracking behaviors of specimens were visually observed. Compressive strengths after exposure to various temperatures were tested. Furthermore, X-ray diffraction (XRD) tests were conducted and micrographs were also taken by means of SEM to illustrate the behaviors in the macroscope.The chemical composition of cement used  is show2), dicalcium silicate (C2S), tricalcium silicate (C3S), calcite (CaCO3), calcium oxide (CaO), and gypsum (CaSO4∙2H2O). It is also noticed that calcium magnesium aluminum oxide silicate (Ca54MgAl2Si16O90), titanium carbide (TiC), and calcium malate (C4H4CaO5) exist in the cement clinker. River sand with a 1.69 fineness modulus and 2.0 mm maximum diameter was utilized. The major chemical, physical, and mechanical properties of PVA fiber used are listed in Sharp characteristic peaks in XRD curves indicate that the main mineral compositions are amorphous silicon dioxide . The mixture proportion and strength properties of ECC containing the above PVA fibers are shown in As can be seen, the water-to-cement ratio (W/C) was 0.45 and the sand-to-cement ratio (S/C) was 0.80. PVA fibers were incorporated in a volume fraction of 2.0%. The flexural strength of such PVE-ECC was evaluated according to ISO 679  as 7.2 ±3 were designed and cast to investigate the behavior of PVA-ECC under ambient temperature, 200, 400, 600, 800, and 1200 °C. Specifically, the test items included surface response, weight loss, compressive strength, composition deterioration behavior, and microstructure change of PVA-ECC. All specimens were prepared in one batch to ensure uniformity. The mixing procedure was consistent with ISO 679 [Six groups in total of 18 cubic specimens of 100 × 100 × 100 mm ISO 679  and prev ISO 679 ,36. AfteAfter 56 days’ curing, the specimens were taken out from the curing chamber for testing. Before heating, they were wiped with dry towels to a state of saturated surface. Then, the cubes were divided into 5 groups and heated in a muffle furnace with the function of programmed control see  to targeThe heating regimes are plotted in The target temperature was kept for 1 h to guarantee a relative thermal stable state for both the furnace and tested specimens. After that, the specimens were cooled down in air to ambient temperature.2θ from 5° to 80° scope in a 0.03° step-length.TGA 4000 thermogravimetry with a temperature accuracy of ±0.1 °C and sensitivity of 0.1 μg was applied to analyze the weight change of the specimen with the increase in temperature. A high-magnification electron microscope was used to observe the surface cracking behavior after heating. The maximum magnifying power was 800×. Furthermore, after residual mechanical properties test, the samples were made into slides and powders with a hammer and triturator. Slides were used to analyze the morphology and microstructure with a JEOL-JSM-7001F  scanning electronic microscope. Meanwhile, the powders were used for XRD analysis with an X-ray diffractometer  with Visual observations of cubic specimens after one-hour exposure to various temperatures are displayed in β-dicalcium silicate and mullite at around 400 to 600 °C for the ultra-lightweight cementitious composite, and hence appear deep red instead. At 800 °C, the cubes appeared light brown. Interestingly, the color turned brownish at 1200 °C  was evaluated as 1.94%, 12.25%, 14.94%, 17.39%, and 21.18% after exposure to temperatures of 200 °C, 400 °C, 600 °C, 800 °C, and 1200 °C, respectively. TG and DTG curves of PVA-ECC also confirmed such weight change, as shown in Herein, evaporation of water may be the main reason for the weight loss below 200 °C. As the temperatures increase up to 400 °C, the loss of chemically bound water and hydrates decomposition (indicated by sharp drop in DTG curves) may further contribute to the mass change of ECC specimens together with the thermal pyrolysis of PVA fibers . Thereafmβ) with heating temperature (T) is provided in Equation (1) for PVA-ECC. A good agreement could be found between the test results and the prediction model. In the absence of actual data, Equation (1) might be applied to empirically predict the remaining weight of PVA-ECC after exposure to elevated temperature.Based on the existing data, the prediction model of mass loss rate  to strength at room temperature (ckf) is defined as the relative compressive strength  to estimate the deterioration rate of post-exposure specimens. The average values of c,T/fckf in the present study are 81.9%, 81.7%, 52.7%, 33.1%, and 10.6% at 200 °C, 400 °C, 600 °C, 800 °C, and 1200 °C, respectively  can be detected at most of the peaks despite the heating temperatures. For CH, no evident change in XRD patterns for 30, 200, 400, and 600 °C was found, but when the heating temperature was elevated up to 800 °C and above, calcium hydroxide was not detected any more. This indicates that the decomposition of CH initiated in the temperature range of 400 °C to 600 °C and finished beyond 600 °C, cf. [3) appeared evidently at 400 °C as a reaction product of C-S-H decomposition under elevated temperature, monohydrocalcite (CaCO3∙H2O) (see XRD curve of 200 °C) decomposition, and calcium oxide (CaO) carbonation. However, CaCO3 was no longer observed at temperatures above 600 °C. Instead, CaO was found beyond 600 °C, which implies that decomposition of CaCO3 initiated at around 500–550 °C under higher pressure conditions in a heated cementitious system. In addition, dicalcium silicate (Ca2SiO4) had been generated when the heating temperature increased to 400 °C and above, the amount of which increased as the temperature further elevated. Hence, the above chemical changes at high temperatures undoubtedly altered cracking behavior and pore structure, consequently leading to the deterioration in macro mechanical strength, especially when the temperature exceeded 800 °C, cf. [As can been seen, C-S-H and CH are clearly observed at the sharp characteristic peaks on the tested intensity–degree curve at ambient temperature, cf. . As the  °C, cf. ,30. By c °C, cf. ,30. Furt2), calcium malate (C4H4CaO5), and magnesium aluminum oxide hydrate (Mg5Al4O11∙5H2O) existed in the tested samples. Herein, TiO2 turned into monohydrocalcite (Ti7O13) at 200 °C, titanium oxide (Ti2O3) at 600 °C, and titanium oxide sulfate hydrate (TiOSO4∙H2O) at 1200 °C, which indicate deoxygenation reaction at high temperature as the ratio of O to Ti decreased from 2:1 at 30 °C to 13:7, 3:2, and 1:1 at 200, 600, and 1200 °C, respectively. C4H4CaO5 turned into calcium malate dehydrate (C4H4CaO5∙2H2O) at 400 °C as a result of moisture evaporation and high temperature, but could be observed beyond 600 °C. As for Mg5Al4O11∙5H2O, it decomposed after a heating of 200 °C, and a more stable reaction product of magnesium carbide (Mg2C3) was found at 800 °C instead. Such variations in compositions of ECC inevitably led to a weaker interfacial transition zone and coarser pore structures. Grossular with rich –OH 3) appeared at 400 °C, and suolunite (Ca2Si2O5(OH)2∙H2O) and hydrobasaluminite (Al2SO4(OH)10∙36H2O) at 800 °C. When the heating temperature was elevated to 1200 °C, calcium sulphate (CaSO4), ferrous sulfite (FeSO3), and srebrodolskite (Ca2Fe2O5) were detected in the XRD curve. Interestingly, brown vuagnatite (CaAlSiO4(OH)) was also found at 1200 °C, which may explain well the apparent color change mentioned in It was also noticed that titanium dioxide  after exposure to high temperatures. The following conclusions can be drawn accordingly:


Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition  or a significant decrease  of 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49–Y51–Q52) and transmembrane domain 2 (G326–K327–F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel. The purinergic P2X receptors P2X1–7) are ATP-gated cation channels that have a widespread distribution in neuronal and non-neuronal cells and are involved in many physiological and pathophysiological processes –7 are AT,4,5,6. P+, K+, and Ca2+ ions, as well as larger organic molecules, such as N-methyl-d-glucamine (NMDG+) [2+ permeability of P2X4 [All P2Xs are organized as trimers . Each su (NMDG+) ,30. Func (NMDG+)  that is  (NMDG+) ,32. The  (NMDG+)  and conf (NMDG+) . The ext (NMDG+) . Several (NMDG+) ,35,36,37 of P2X4 . Analysi of P2X4 . Previou of P2X4 . These rHere, we tested a hypothesis that the conserved residues in the extracellular vestibule and the nonconserved residue F322  are important for P2X7 function. We substituted these residues with an alanine residue and characterized the functional properties of these mutations after expression in human embryonic kidney 293T (HEK293T) cells. The results show that clusters of conserved residues proximal to TM1 and TM2 are important for receptor trafficking, structure, and gating and that P2X7 function is critically dependent on the properties of the nonconserved residue F322 at the top of the extracellular vestibule and between the extracellular and central vestibules.The extracellular vestibule of rat P2X7 (rP2X7) is formed by segments A44–I58 and F322–Y336 that are proximal to TM1 and TM2, respectively A. StructThe wild-type (WT) rP2X7 and alanine mutants of these residues were expressed in HEK293T cells, and the receptor function was examined using patch-clamp whole-cell recordings and dye uptake measurements. We used different concentrations of 2′,3′-O-(benzoyl-4-benzoyl)-ATP  to investigate the effect of mutagenesis on receptor activity and sensitivity to agonists C.1), the secondary current growth (I2) produced by prolonged (40–60 s) exposure to the agonist, and the amplitude of the maximum current (I1 + I2) induced by application of 300 µM BzATP after current facilitation  on average by 1.5 ± 0.2-fold  after prolonged exposure to the agonist, and the EC50 decreased significantly (p < 0.01) from 45 ± 4.6 µM to 20 ± 2 µM  reduced maximum responses were K49A, G326A, K327A, and F328A (p < 0.05) changes in EC50 values compared to that of the WT receptor  was not significantly different for the F322A mutant; however, this receptor showed significantly (p < 0.01) increased I1 (p < 0.01) decrease in the EC50 value  reduced amplitude of BzATP-induced current  to test a hypothesis that this effect reflects changes in the level of receptors on the cell surface. We examined membrane fractions of total protein derived from HEK293T cells expressing the WT or a mutant receptor (p < 0.05) reduced expression in the plasma membrane fraction, whereas other mutants (K49A and G326A) were present in variable quantities  using confocal microscopy C. These Taken together, these results suggest that for the Y51A, Q52A, and F328A mutants, the loss of receptor function reflects the lack of their expression at the plasma membrane, while altered function of K49A, G326A, and possibly G323A is due to changes in the intrinsic channel properties.1 + I2 = 2.6 ± 0.3 nA; K49A, I1 + I2 = 1.0 ± 0.2 nA; p < 0.01; K49R, I1 + I2 = 3.1 ± 0.4 nA; p > 0.05; 50 = 20 ± 2 µM; K49A, EC50 = 63 ± 26 µM; p < 0.05; K49R, EC50 = 36.2 ± 12 µM; p > 0.05; In contrast to the K49A mutant, the K49R mutant A did notThese data showed that the positive charge at position 49 is important for P2X7 channel gating.p < 0.01) reduced maximum current amplitude  could not rescue the receptor function .These results show that a large aromatic residue at position 51 is essential for receptor trafficking and function.1 + I2 compared with that of the WT receptor , but the maximum amplitude of the initial BzATP-induced current in naïve cells was significantly (p < 0.05) higher in the F322A receptor than in the WT receptor  dramatically increased by 60-fold compared with that in the WT receptor  increased sensitivity of the F322A receptor to agonists  or a polar aromatic residue (F322Y) rescued the slow current facilitation and fast deactivation  and recop < 0.01) increased by approximately 3-fold, and this effect could also be reversed to various degrees by substituting this residue with nonpolar or polar hydrophobic residues  did not generate functional receptors .Together, these data indicate that the hydrophobicity and size, rather than aromaticity, of the residue at position 322 are important for P2X7 gating to a closed state. Additionally, these data also show that nearby conserved glycine at position 323 is essential for P2X7 function and probably operates as a hinge when the receptor undergoes activation-induced conformational changes.p < 0.01) reduced by ~90% in cells expressing the K49A, Y51F, Y51W, G326A, K327A. and F328A mutants (p < 0.01) in cells expressing the F322A, F322L, F322W, and F322Y mutants  and TM2 (G326–K327–F328) domains are important for membrane expression, receptor structure, and gating properties and that the nonconserved residue (F322), which is unique for P2X7 and P2X4, dramatically controls the affinity of the receptor for its ligands, deactivation kinetics, and dye uptake ability. 50 compared to that of the WT receptor [We found here that the maximum current amplitude and receptor sensitivity to BzATP were significantly reduced for the K49A mutant compared to those for the WT receptor. In contrast, this mutant at an equivalent position in P2X4 shows no significant difference in the current amplitude or ECreceptor , indicatreceptor ,40,47. Ireceptor ,49, sugg50 values and the hydrophobicity (Substitutions of the nonconserved residue (F322), which is present only in P2X7 and P2X4, at the top of the extracellular vestibule and between the extracellular and central vestibules showed an enhanced facilitation of current kinetics, an accelerated dye uptake, an increased affinity for its ligands, and a slower rate of deactivation, indicating that this residue most likely controls gating and intra-subunit signal transduction from the extracellular agonist-binding site to the transmembrane domain. In contrast, alanine or cysteine substitutions of phenylalanine at equivalent positions in P2X4 respond to ATP with substantially reduced (approximately 10-fold) maximum current amplitude and exhibit only approximately 2-fold higher sensitivity to agonists , suggesthobicity C or voluhobicity D of the Although the nearby G323 residue is conserved among the P2X receptor subtypes, its critical functional importance is shown only for P2X4  and P2X7p < 0.05) sensitivity to agonists. The dye uptake ability of cells expressing these mutants being inhibited by ~90% thus must be due to the reduced density of receptors in the membrane, which was confirmed for the F328A mutant. Our results show that both reduced membrane expression  and lower receptor sensitivity to agonists (K49A and Y51F) abolish dye uptake ability, while increased receptor sensitivity (F322 mutants) results in accelerated dye uptake by cells expressing these mutant receptors. Overall, the results of previous studies [The G326A, K327A, and F328A mutants exhibited significantly reduced (by ~60–80%) amplitudes of agonist-evoked currents without changes in receptor sensitivity to agonists, except G326A, which showed slightly enhanced  and TM2 (G326–K327–F328) are important for receptor trafficking and structure and thus have functions similar to those observed previously in the P2X4 receptor . The mos2 atmosphere at 37 °C. The cells were cultured in 75 cm2 plastic culture flasks  for 36–72 h until they reached 80–95% confluence. On the day before transfection, ~150,000 cells were seeded on 35 mm culture dishes  and incubated at 37 °C for at least 24 h. Transfection was performed using 2 μg of DNA and 2 μL of jetPRIME reagent in 2 mL of Dulbecco’s modified Eagle’s medium according to the manufacturer’s instructions . After 24 h of incubation, the transfected cells were mechanically dispersed and reseeded on 35 mm Corning 3294 CellBIND Surface cell culture dishes  for 2–8 h prior to recording. For the dye uptake measurements, the transfected cells were plated on 12 mm poly-L-lysine-coated coverslips .Experiments were performed using HEK293T cells , which were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum , 50 U/mL penicillin, and 50 μg/mL streptomycin  in a humidified 5% CORat full-length rP2X7 receptor cDNA subcloned into the biscistronic enhanced fluorescent protein expression vector pIRES2-EGFP was a kind gift from Dr. S.S. Stojilkovic, NICHD/NIH, Bethesda, MD, USA. For constructing the YFP C-terminal-tagged rP2X7, full-length rP2X7 was first amplified by PCR using a 3′ oligonucleotide primer (Eurofins) designed to replace the stop codon by an in Frame Xho1 restriction site as previously described for other P2X subtypes ,55. The 2, 1 mM MgCl2, 10 mM HEPES, and 10 mM D-glucose and adjusted to pH 7.3 using 1 M NaOH . All solutions with agonists were prepared on the day of experiment and were applied using the RSC-200 Rapid Solution Changer . Sensitivity of mutated receptors was measured using a short (2–5 s) application of 3–300 µM 2′,3′-O-(benzoyl-4-benzoyl)-ATP , an artificial but potent agonist of the P2X7 receptor [50 values of naïve (not previously stimulated) cells, the responses from 15–50 different naïve cells were pooled, and dose–response curves were constructed using the mean values.Currents were recorded in a whole-cell configuration using an Axopatch 200B patch-clamp amplifier . All current recordings were captured and stored using the pClamp 9.0 software package in conjunction with the Digidata 1322A A/D converter . The experiments were performed on single cells with an average capacitance of 10 pF, and the membrane potential was held at –60 mV. Patch electrodes were filled with solution containing 145 mM NaCl, 10 mM EGTA, and 10 mM HEPES; the pH was adjusted using 10 M NaOH to 7.3 . The osmreceptor . To estiStudies on the cellular accumulation of EtBr were performed using an epifluorescent microscope . Transfected HEK-293T cells plated on glass coverslips were bathed in normal extracellular solution, imaged using the 40× water immersion objective at room temperature (20–25 °C), and identified by the fluorescence signal of green fluorescein protein. EtBr (5 μM) and BzATP (100 μM) in normal extracellular solution were added using a fast gravity-driven microperfusion system made in our laboratory and consisting of nine glass tubes, each approximately 400 μm in diameter, with a common outlet of approximately 300 μm in diameter. The application tip was routinely positioned at about a 500 μm distance from the recorded cells and approximately 50 μm above the surface of the coverslip. Change in fluorescence was recorded using MicroMAX CCD camera . Hardware control and image analysis were performed using MetaFluor software . EtBr was excited at 526 nm, and emission light was collected at 605 nm.. The average fluorescence signal of 10 cells was analyzed on each coverslip and each experiment was repeated 3–7 times. The rate of dye uptake was determined as the amplitude of EtBr signal after 100 s of agonist application.2 and 1 mM MgCl2 for 1 h at 4 °C. Nonreacted sulfo-NHS-SS-biotin was quenched by washing cells with cold PBS, pH 8.2, containing 100 mM glycine, 0.1 mM CaCl2, and 1 mM MgCl2. Cells were harvested in lysis buffer  Nonidet P-40, 1% (v/v) SDS and protease inhibitor ) before gentle sonication and centrifugation at 14,000× g for 10 min at 4 °C. Biotinylated proteins were pulled down from equivalent amounts (~1 mg) of “total” protein (whole cell lysate) with 50 µL of neutravidin agarose beads  and eluted into 50 µL of Laemmli sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer supplemented with 50 mM dithiothreitol. To determine the amounts of biotinylated P2X7 (membrane) or “total” P2X7 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, 5 µL of eluted protein and equivalent amounts (~50 μg) of “total” protein were separated on 4–15% precast gels , followed by Western blot with anti-P2X7 monoclonal  or GAPDH , antibody, and detected using horseradish peroxidase coupled anti-rabbit or anti-goat Fc-IgG antibody at dilutions of 1:1000 and 1:10,000, respectively. The chemiluminescent signals were captured using an Image Analyzer , and signal intensity (arbitrary densitometric units) was quantified using Image J software . The relative surface P2X7 expression was calculated as the ratio of membrane to the total P2X7 protein after normalization against GAPDH.Cell-surface proteins were biotinylated using EZ-Link sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithio-propionate  following established protocol ,57. BrieTransiently transfected HEK293T cells with YFP-tagged rP2X7-WT receptor and its mutants  were plated onto 12 mm poly-L-lysine -coated coverslips and cultured in Opti-MEM  supplemented with 5% fetal calf serum in a water-saturated atmosphere of 5% CO2 and 95% air at 37 °C. The following day, cells were fixed in ice-cold 4% formaldehyde, pH 7.4, in phosphate-buffered saline for 15 min and marked by wheat germ agglutinin conjugated with Alexa633 . After washing residual agglutinin by PBS, coverslips were mounted in Aqua/Poly-Mount medium  onto glass slides. The localization of YFP-tagged receptors in cells was examined by laser scanning confocal microscopy . Images were collected under a 63× objective lens with further zoom applied.max/(1 + (EC50/x)h), where y is the amplitude of the current evoked by BzATP, Imax is the maximum current amplitude induced by 300 μM BzATP, EC50 is the agonist concentration producing 50% of the maximal response, h is the Hill coefficient, and x is the concentration of BzATP . Hill coefficient was fixed to 1.3 in all experiments, and a value obtained for the P2X7 WT by fitting. The kinetics of deactivation (current decay evoked by washout of 30 or 100 μM BzATP in naïve (not previously stimulated) cells) were fitted by a single exponential function (y = A1 exp(−t/τ)) or by the sum of two exponentials (y = A1 exp(−t/τ1) + A2 exp(−t/τ2)) using the program pClamp 10 (Molecular Devices), where A1 and A2 are relative amplitudes of the first and second exponentials, and τ1 and τ2 are the time constants. Weight time constant was calculated as y = ((A1τ1) + (A2τ2))/(A1+A2). The derived time constants for deactivation were labeled as τoff.The concentration–response data points were fitted with the equation y = Ip < 0.01 and * p < 0.05) in SigmaStat 2000 v9.0, for comparison to a single control.All numerical values in the text are reported as means ± SEMs. Comparisons between two groups were performed by Student’s t-test. Statistical comparison of multiple groups was made by using one-way ANOVA followed by SNK or Dunnett’s test (**


A major risk factor promoting tumor development is chronic inflammation and the use of nonsteroidal anti-inflammatory drugs (NSAID), including ibuprofen, can decrease the risk of developing various types of cancer, including colorectal cancer (CRC). Although the molecular mechanism behind the antitumor properties of NSAIDs has been largely attributed to inhibition of cyclooxygenases (COXs), several studies have shown that the chemopreventive properties of ibuprofen also involve multiple COX-independent effects. One example is its ability to inhibit the alternative splicing event generating RAC1B, which is overexpressed in a specific subset of BRAF-mutated colorectal tumors and sustains cell survival. Here we describe the mechanism by which ibuprofen prevents RAC1B alternative splicing in a BRAF mutant CRC cell line: it leads to decreased translocation of SRPK1 and SRSF1 to the nucleus and is regulated by a WNK1/GSK3β/SRPK1 protein kinase complex. Surprisingly, we demonstrate that ibuprofen does not inhibit the activity of any of the involved kinases but rather promotes disassembly of this regulatory complex, exposing GSK3β serine 9 to inhibitory phosphorylation, namely by AKT, which results in nuclear exclusion of SRPK1 and SRSF1 hypophosphorylation. The data shed new light on the biochemical mechanisms behind ibuprofen’s action on alternative spliced RAC1B and may support its use in personalized approaches to CRC therapy or chemoprevention regimens. Cancer is the second leading cause of death globally  and one Here we unveiled the molecular mechanisms of how ibuprofen interferes with alternative splicing in BRAF mutant CRC cells. We show that ibuprofen disrupts a signal transduction pathway by, unexpectedly, interfering with the assembly of a protein kinase complex, composed by WNK1, GSK3β and SRPK1. This leads to changes in the subcellular localization of splicing factor SRSF1, which promotes inclusion of exon 3b into the mRNA and subsequent expression of RAC1B.Previously, we described that the anti-inflammatory drug ibuprofen inhibited RAC1B expression in HT29 colorectal tumor cells , but thein vitro kinase assay to measure the activity of recombinant SRPK1 towards the commonly used synthetic RSpeptide (Abcam) substrate. We observed that, in contrast to the highly selective, ATP-competitive SRPK inhibitor SRPIN340 . The cytosolic fraction was collected by centrifuging the lysate at 6000 rpm for 4 min (at 4°C) and adding the supernatant to 60 μl of 5× Laemmli SDS sample buffer. The pellet containing the nuclear fraction was washed once in cytosol buffer and then resuspended in 75 μl of nuclear buffer . The resuspended nuclear fraction was incubated on ice during 20 min  and collected by centrifuging the lysate at 10000 rpm for 10 min (at 4°C) and adding the supernatant to 15 μl of 5× Laemmli SDS sample buffer. Equal volumes of both fractions were analyzed side by side on Western blots. Results were confirmed in at least three independent experiments.2 and 1 mM MgCl2). Cells were then incubated with the crosslink solution DSP+SPSP  for 2 h. To quench the reaction 50 mM Tris/HCl pH 8.0 was added for 15 min. Cells were again washed 3× with cold PBS-CM and lysed in 750 μL lysis buffer  glycerol, 1% (v/v) NP40) supplemented with a protease inhibitor cocktail composed of 1 mM PMSF, 1 mM 1,10-phenanthroline, 1 mM EGTA, 10 μM E64, and 10 μg/mL of each aprotinin, leupeptin, and pepstatin A . Cell lysates were cleared at 3000× g for 5 min at 4°C. The supernatant was pre-cleared by adding 45 μL streptavidin-agarose beads (Sigma-Aldrich), rotated for 1 h at 4°C and centrifuged for 1 min at 3000× g. Approximately 700 μL of the pre-cleared lysates were recovered and added to the beads prepared as described in the following section (see Beads preparation) and rotated overnight at 4°C. After incubation, the tubes were centrifuged for 1 min at 3000× g and washed 6× in cold wash buffer . Captured proteins were recovered in 50 μL 2× SDS modified sample buffer with extra DTT (100 mM) and analyzed by SDS-PAGE and Western blotting as described in the corresponding section.HT29 cells were seeded in 100 mm culture dishes at near 100% confluence, placed on ice and washed three times with ice cold PBS-CM (PBS pH 8.0 containing 1 mM CaCl60 μl of dry G-protein agarose beads (Roche) were incubated with 1 μg of mouse anti-SRPK1 (BD Transduction Laboratories) or 1.5 μg of sheep anti-WNK1 antibody (Dundee University) diluted in PBS for 1 h at 4°C. Beads were washed 3× with PBS and crosslinked to the antibody with 4% (v/v) formaldehyde in PBS for 1 h at 4°C. After crosslink, beads were washed 3× with PBS and blocked with 2% BSA in PBS solution rotating for 1 h at 4°C, washed 3× with PBS and added to the pre-cleared lysate recovered as described in the Immunoprecipitation section.Cells were grown on 10 × 10 mm glass coverslips to about 20–30% confluency, incubated and transfected as indicated above, then washed twice in PBS, immediately fixed with 4% (v/v) formaldehyde in PBS for 60 min at room temperature, and subsequently permeabilized with 0.5% (v/v) Triton X-100 in PBS for 30 min at room temperature. Cells were then labeled for 60 min with either a 1:500 dilution of SRPK1 antibody (BD Transduction Laboratories) or a 1:100 dilution of SRSF1 antibody (anti-SF2/ASF (96) (sc-33652) from Santa Cruz Biotechnology), combined with a 1:250 dilution of HA antibody (Sigma-Aldrich) for HA-GSK3βS9A transfected cells, washed 3 times in PBS for 5 minutes with gentle shaking, followed by 30 minutes incubation with a 1:250 dilution of Alexa Fluor 488 and rabbit Alexa Fluor 546 (both from Invitrogen). Coverslips were washed 3 times in PBS, briefly stained with 0.5 ng/ml DAPI (Sigma), washed again and then mounted in VectaShield (Vector Laboratories) and sealed with nail polish. Images were recorded with the 405 nm, 488 nm and 546 nm laser lines of a Leica TCS-SPE confocal microscope and processed with Adobe Photoshop software.2, 3 mM MnCl2, 3 μM Na2VO4, 1.2 mM DTT and 0.05 mg/ml BSA. After 1 h of incubation at 30°C, 10 μL of ADP-Glo reagent was added and incubated for 40 min at room temperature. Then, 20 μL of kinase detection reagent was added and, after an incubation time of 30 min, luminescence was measured on a GloMax® 96 microplate luminometer (Promega). All the kinases used were purchased from ProQinase. For WNK1 20 ng of kinase, 3.25 μg of RS Repeat peptide (Abcam) and 100 μM ATP were used, for SRPK1 50 ng of kinase, 1.5 μg of RS Repeat peptide (Abcam) and 2 μM ATP. For each kinase reaction, two control reactions were made: one without substrate and other without kinase. 500 μM ibuprofen (Sigma-Aldrich) was added to the reactions, using DMSO as control. Specific inhibitors for each kinase were 10 μM WNK463 (Carbosynth) for WNK1, and 25 μM SRPIN340 (kindly provided by Dr. Masatoshi Hagiwara) for SRPK1.This assay is more sensitive for measuring low to moderate protein kinase activities as it amplifies in an enzymatic conversion step the levels of ADP that result from the phospho-transfer. ADP-Glo™ Kinase Assay kit was purchased from Promega. The primary reaction consisted of the kinase, substrate and ATP in 60 mM HEPES, pH 7.5, 3 mM MgCl® Luminescent Kinase Assay kit was purchased from Promega. The primary reaction consisted of the kinase, substrate and ATP in 60 mM HEPES, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na2VO4, 1.2 mM DTT and 0.05 mg/ml BSA. After 1 h of incubation at 30°C, 20 μL of Kinase-Glo® Reagent was added and mixed at room temperature for 10 min, and luminescence was measured on a GloMax® 96 microplate luminometer (Promega). All the kinases used were purchased from ProQinase. For GSK3β we used 140 ng of kinase, 1 μg of GSM (Millipore), or 1 μg of SRPK1, and 1 μM ATP. For each kinase reaction, control reactions were made without substrate or without kinase. 500 μM ibuprofen (Sigma-Aldrich) was added to the reactions, using DMSO as control. As specific inhibitor for GSK3β we used 10 μM CHIR99021 (BioVision). Note that all the above conditions included also 25 μM SRPIN340 (kindly provided by Dr. Masatoshi Hagiwara) in order to inhibit SRPK1 kinase activity.This assay is more accurate for measuring higher protein kinase activities as it directly measures the consumption of co-factor ATP during the phospho-transfer. Kinase-Glot-tests for paired samples or ANOVA tests followed by post-hoc Tukey’s tests when comparing multiple treatments. P < 0.05 was accepted as the level of statistical significance. Shown data reflect the mean ± SEM from at least three independent experiments.Data were analyzed using Student’s


Plasmodium merozoite surface protein 4 (MSP4) has been listed as a blood-stage subunit vaccine candidate for malaria. Infection with Plasmodium ovale species including P. ovale wallikeri and P. ovale curtisi, is also a source of malaria burden in tropical regions where it is sometimes mixed with other Plasmodium species. However, little is known about P. ovale MSP4.There is an urgent need for an effective vaccine to control and eradicate malaria, one of the most serious global infectious diseases. msp4 gene was amplified through polymerase chain reaction using genomic DNA extracted from blood samples of 46 patients infected with P. ovale spp. and amplified products were sequenced. Open reading frames predicted as immunogenic peptides consisting of 119 and 97 amino acids of P. ovale curtisi MSP4 (PocMSP4) and P. ovale wallikeri MSP4 (PowMSP4), respectively, were selected for protein expression. Recombinant proteins (rPoMSP4) were expressed in Escherichia coli, purified, analysed, and immunized in BALB/c mice. The specificity of anti-MSP4-immunoglobulin (Ig) G antibodies was evaluated by Western blot and enzyme-linked immunosorbent assays, and cellular immune responses were analysed via lymphocyte proliferation assays.The P. o. wallikeri isolate. Further, truncated PoMSP4 segments were successfully expressed and purified as ~ 32 kDa proteins. Importantly, high antibody responses with end-point titres ranging from 1:10,000 to 1:2,560,000 in all immunized mouse groups were observed, with high IgG avidity to PocMSP4 (80.5%) and PowMSP4 (92.3%). Furthermore, rPocMSP4 and rPowMSP4 cross-reacted with anti-PowMSP4-specific or anti-PocMSP4-specific antibodies. Additionally, anti-PoMSP4 IgG antibodies showed broad immuno-specificity in reacting against rPoMSP1 and rPoAMA1. Lastly, PocMSP4- and PowMSP4-immunized mice induced cellular immune responses with PocMSP4 (36%) and PowMSP4 cells (15.8%) during splenocyte proliferation assays.Full peptide sequences of PocMSP4 and PowMSP4 were completely conserved in all clinical isolates, except in the epidermal growth factor-like domain at the carboxyl terminus where only one mutation was observed in one Findings from this study suggest conservation in PoMSP4 protein sequences and high immunogenicity was observed in rPoMSP4. Furthermore, induction of immune responses in PocMSP4- and PowMSP4-immunized mice informed that both humoral and cellular immune responses play crucial roles for PoMSP4 in protection. Plasmodium falciparum  and full length of PoAMA1 [pocama1 (PlasmoDB: PocGH01_09039800) and powama1 (GenBank: SBT36045.1)] merozoite surface proteins, which were previously expressed and preserved in our laboratory, were used for specificity tests of PoMSP4 protein-raised antibodies.The N-terminal of PoMSP1  and lysed by sonication. The insoluble fraction was separated by centrifugation at 15,000×g for 15 min at 4 °C. The soluble fraction was applied to a column containing 1.0 mL of Ni-nitrilotriacetic acid-agarose (Qiagen) and then washed with 10 mL of purification buffer containing 20 mM imidazole. Recombinant proteins were eluted from the column with purification buffer containing 250 mM imidazole and then exchanged into Tris–HCl storage buffer  using a 30 kDa ultrafiltration tube (Millipore). Proteins were stored at − 80 °C until use.The concentration of recombinant proteins (rPoMSP4) was determined through the Bradford method using bovine serum albumin (BSA) as standard . Purified proteins were analysed by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining (Beyotime Biotech) to assess the expression level and immunoreactivity. The separated proteins from SDS-PAGE were electrophorectically transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon) and blocked overnight in Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% skimmed milk at 4 °C. The membranes were probed with anti-His antibody  at 1:5000 dilution along with primary antibody dilution buffer (Meilunbio) overnight at 4 °C. Membranes were washed three times with 0.1% TBST and treated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cowin Biotech) at 1:5000 dilution for 90 min. Finally, the membranes were analysed with a ChemiDoc MP imaging system (Bio-Rad).Six- to eight-week-old female BALB/c mice were used for immunizations as follows. Mice were grouped into the rPoMSP4-immunized (n = 5 per group) and negative control groups (n = 3 per group). Each mouse was intraperitoneally injected with 50 µg of rPocMSP4, rPowMSP4, or PBS, all of which were diluted in PBS with complete Freund’s adjuvant (Sigma). An equal volume of antigen with incomplete Freund’s adjuvant (Sigma) was used for subsequent boosters, which were administered on days 21 and 42 post-immunization intraperitoneally. The control group was administered an equal amount of PBS and adjuvant. Mouse blood samples were collected from the tip of the tail on days 0, 7, 14, 28, 35, and 49. Sera were obtained via centrifugation for 20 min at 2000 × rpm and stored at − 80 °C.Purified rPoMSP4 was tested against sera from rPocMSP4- or rPowMSP4-infected mice to assess anti-PoMSP4 IgG antibodies through Western blot analysis. During the assays, PVDF membranes were incubated with antisera (1:2000 dilutions) from the rPoMSP4-immunized group or negative control group, followed by HRP-conjugated goat anti-mouse IgG (Cowin Biotech) at 1:5000 dilution.2SO4 was added to stop the reaction. The absorbance at OD of 450 nm was measured using a microplate reader . Furthermore, anti-PoMSP4 IgG antibodies were tested against rPoMSP1 and rPoAMA1 antigens via ELISA to test for the specificity of antibodies.Levels of IgG antibodies targeting PoMSP4 in mouse sera were detected via enzyme-linked immunosorbent assays (ELISA). In brief, 96-well ELISA plates were coated with 50 ng of rPoMSP4 antigen dissolved in coating buffer solution (15 mM sodium carbonate and 35 mM sodium bicarbonate in distilled water) overnight at 4 °C. After three times wash with PBS containing 0.1% of Tween-20 (PBST), the plates were blocked with 1% BSA in PBS and incubated at room temperature for 2 h. Thereafter, individual mouse sera (100 µL) diluted at different dilutions were added on the plate and incubated at room temperature for 2 h. The plates were washed again three times with PBST and HRP-conjugated goat anti-mouse IgG antibodies (Southern Biotech) at 1:5000 dilution and incubated for 1 h 30 min at room temperature. The plates were finally washed three times with PBST and incubated with 3,3′,5,5′-tetramethylbenzidine (Invitrogen) substrate for a few minutes in the dark, and 2 M H5 cells/well of PoMSP4- and PBS-immunized cells was treated with 10 µL of PocMSP4 (5 µg/mL), 10 µL of PowMSP4 (5 µg/mL), or 10 µL of concanavalin A , as positive control, in 96-well flat-bottom microtitre plates and then incubated for 72 h at 37 °C with 5% CO2. Thereafter, 10 µL of CCK-8 was added to each well, and the plates were incubated for 2 h at 37 °C and measured at 450 nm using a microplate reader.Lymphocyte proliferation was measured using a cell counting kit-8 . A certain amount of 5 × 10pomsp4 genes from all clinical isolates were translated to the deduced amino acid sequences using the MegAlign module of Lasergene 7 software package (DNAstar) and then aligned with reference sequences to assess conservation within PoMSP4. Amino acid sequences of the segments of PocMSP4 and PowMSP4 isolates were aligned with those of the PocGH01_04023000 and LT594508.1 reference strains, respectively. Sequence alignment for all P. ovale isolates was performed using MEGA v.7.0 software.Full nucleotide sequences of P) value of < 0.05 indicated a significant difference.Statistical analysis and graphing were conducted using GraphPad Prism software version 5.0 . SPSS v.16.0 was performed to analyse cross-reaction and antibody responses. Student’s t-test with probability ; however, this single mutation could not affect the antigenicity of MSP4 proteins because EGF domains have shown relatively poor immunogenicity [The full length of ity Fig. . The msprthologs , 33, 34.genicity , 31, 37.pomsp4 fragments were successfully amplified in isolates through PCR, generating single PCR products with the expected size of 360 bp for pocmsp4 and 294 bp for powmsp4. Direct sequencing of purified PCR fragments showed no superimposed signal on the electropherograms for pomsp4  stimulations. Results showed that rPoMSP4-induced cell proliferation was at 36% for PocMSP4 and 15.8% for rPowMSP4 . However, significant differences in cross-reaction were detected between rPocMSP1 to anti-rPocMSP4 sera and rPocMSP4 to anti-rPocMSP4 sera (p < 0.0001); whereas no significant difference was observed between rPocMSP4 and PowMSP1 to anti-rPocMSP4 sera , as well as between rPowMSP4 and PowMSP1 to anti-rPowMSP4 sera  were completely conserved. These results were in accordance with other reports that showed a high degree of conservation of msp4 orthologs in P. falciparum isolates [pomsp4 can be useful in PCR-based diagnostic testing for P. ovale spp.The effectiveness of malaria vaccines mostly depends on the high degree of conservation and excellent level of immunogenicity. The present study analysed 46 isolates , 34, 37.The development of blood-stage vaccines against malaria focuses on surface-exposed proteins of erythrocytes infected with the malaria parasites, which are accessible for antibodies. Thus, humoral immune responses are important against malaria parasites because they play essential roles in protecting against blood-stage malaria , 39. AntLymphocytes play an important role in the immune system because these cells determine the specificity of the immune response to infectious microorganisms and other foreign substances. Lymphocyte proliferation assays are widely used to measure cell-mediated T-cell immunity and responses to specific antigens , 45. BesP. ovale that were used in this study may be more widely evaluated as potential vaccine candidates.Immunization with conserved rPoMSP4 protein fragments resulted in a remarkable humoral immune response. Cross-reaction between rPocMSP4 and rPowMSP4 proteins or between rPoAMA1 and rPoMSP1 proteins to anti-rPoMSP4 was detected, suggesting that these proteins shared similar antigenic determinants. PoMSP4 could induce cellular immune responses. Though future studies are needed to assess the mechanism by which PoMSP4 fragments play roles in immune protection, but the fragments of Additional file 1: Table S1. Information on imported Plasmodium ovale curtisi and Plasmodium ovale wallikeri used in this study.Additional file 2: Table S2. The msp4 Gene ID number of other Plasmodium species.Additional file 3: Figure S1. Alignments for the amino acid sequences of Plasmodium ovale curtisi and Plasmodium ovale wallikeri MSP4 in all amplified clinical isolates a. PocMSP4 amino acid sequences b. PowMSP4 amino acid sequences.