CP-AMPARs in the nucleus accumbens (NAc) mediate cue-triggered motivation for food and cocaine. In addition, increases in NAc CP-AMPAR expression and function can be induced by cocaine or sugary, fatty junk-foods. However, the precise nature of these alterations and the degree to which they rely on the same underlying mechanisms is not well understood. This has important implications for understanding adaptive vs. maladaptive plasticity that drives food- and drug-seeking behaviors. Furthermore, effects of junk-foods on glutamatergic plasticity in females are unknown. Here, we use a combination of protein biochemistry and whole-cell patch clamping to determine effects of diet manipulation on glutamatergic plasticity within the NAc of males and females. We found that junk-food consumption increases silent synapses and subsequently increases CP-AMPAR levels in males in the NAc of male rats. In addition, a brief period of junk-food deprivation is needed for the synaptic insertion of CP-AMPARs and the maturation of silent synapses in males. In contrast, junk-food did not induce AMPAR plasticity in females but may instead alter NMDAR-mediated transmission. Thus, these studies reveal sex differences in the effects of junk-food on NAc synaptic plasticity. In addition, they provide novel insights into how essential food rewards alter NAc function. Activity in mesocorticolimbic circuits influences a range of motivational processes including the pursuit of food and drug rewards. There is general agreement that the nucleus accumbens (NAc) is a central hub in this network, integrating information from glutamatergic and dopaminergic inputs to direct behavior toward essential rewards like food and sex, as well as nonessential rewards like cocaine –3. WhileGlutamatergic plasticity within the NAc core is a key mediator of enhanced motivation for both food and cocaine –10. For Increases in NAc core CP-AMPAR synaptic transmission mediate the incubation of cocaine craving, are persistent (lasting months), and require a relatively long drug free period (~3 weeks). During this drug free period, there is an accumulation of CP-AMPARs on the cell surface that is accompanied by their synaptic recruitment and retention , 15–17. On the other hand, consumption of JF for as few as 10 days increases NAc core CP-AMPAR synaptic transmission in males after just 1 day of JF deprivation, an effect that persists for at least 1 month . Howeverhttps://sites.google.com/a/umich.edu/ferrario-lab-public-protocols/ for additional details. Ns for all data are given in the “Results” section below. In scientific papers “N” is a common abbreviation for the number of subjects in a a study. Example, “10 people were in the control group, and 10 people were in the experimental group, thus the N was 10 per group”.Male and female selectively bred obesity-prone and obesity-resistant rats were used. The original selection was conducted by Barry Levin from outbred Sprague Dawley rats . Rats usControl groups were given free access to standard lab chow  in their home cage, whereas experimental groups were given free access to a JF diet made in house , 24. The3-crosslinking procedure followed by western blotting to determine the surface and intracellular expression of GluA1 and GluA2 AMPAR subunits ; pH 7.4) and homogenized by sonication. Samples were then stored at −80 °C. For SDS-PAGE and western blotting samples were heated  in Laemmli sample treatment buffer with 5% β-mercaptoethanol, loaded (20 µg protein), and electrophoresed on 6% gels made in house. Proteins were transferred onto PVDF membranes . Membranes were rinsed, blocked , and incubated overnight (4 °C) with primary antibodies to GluA1  or GluA2 . Membranes were then washed in TBS-T, incubated with HRP-conjugated secondary , washed, and immersed in chemiluminescence detecting substrate . Images were acquired on film and Ponceau S (Sigma-Aldrich) was used to determine total protein in each lane. Bands of interest were then quantified using Image J (NIH).We first determined the effect of 10 days of JF followed by 2 weeks of JF deprivation on AMPAR subunit surface expression. During the deprivation period, rats in the JF group were given free access to standard lab chow and JF was no longer available. Next, we used an established BSsubunits , 13, 25*n = 26) and obesity-resistant (n = 23) male rats were separated into three groups: chow fed, JF no deprivation, and JF 24-h deprivation. The JF deprivation and no deprivation groups received JF for 10 days, while the chow group remained on chow throughout the study. The JF deprivation group received chow diet for 24 h before tissue collection and BS3-crosslinking, whereas the JF no deprivation remained on JF throughout. GluA1 and GluA2 surface expression were determined as described above.To determine the effects of JF deprivation on GluA1 and GluA2 surface expression, a separate cohort of obesity-prone  from obesity-prone male rats. Recordings were made from chow controls, and on day 10 of JF exposure or after 24–48 h of JF deprivation. This timing was chosen to improve feasibility of recordings, rather than timing all recordings to 24 h. Recordings from chow controls and JF groups were interspersed. MSNs were identified visually by cell body size (~15 μm in diameter) and by their capacitance and resting membrane potential after break in, as required recording conditions prevent the proper evaluation of membrane properties distinct to MSNs.A receptor antagonist picrotoxin (50 µM). For measures of the AMPA/NMDA ratio, eEPSCs were recorded at +40 and −70 mV before and after bath application of NMDAR antagonist APV as previously described ..A receptFor silent synapse measures, recording conditions and solutions were as previously described , 30. Pip3-crosslinking procedure described above. Separate cohorts with their own chow control groups were used for 10 days and 30 days studies. These durations and time points were chosen because they produce increases in GluA1 surface expression in males [Studies in females were conducted alongside those in males but are described separately for the sake of clarity. We first determined the effects of 10 or 30 days of JF diet consumption followed by 2 weeks of JF deprivation on GluA1 and GluA2 surface expression using the BSin males  and resuSeparate cohorts of obesity-prone female rats were used to examine potential effects of 10 days of JF followed by 2 weeks of JF deprivation on CP-AMPARs and AMPA/NMDA ratio using the electrophysiological approaches described above. All recordings were conducted from slices prepared when females were in the metestrus/diestrus phase of the cycle. This phase was chosen because this is when motivation for food, food intake, and cue-triggered food seeking are highest in females –34. Estrt-tests, one-way or two-way ANOVAs, and Sidak’s post-hoc multiple comparisons were used . Electrophysiology data were analyzed using Clampfit 10.4 (Molecular Devices). Comparisons were made between data collected within the same cohort of animals .Two-tailed Weight gain and food intake analysis are summarized in Supplementary Tables In females destined for biochemical studies, obesity-prone rats gained more weight than obesity-resistant rats regardless of diet manipulation (Supplementary Table n = 10 rats per group). GluA1 surface expression was increased in obesity-prone but not obesity-resistant rats following 2 weeks of JF deprivation  = 16.1, p = 0.0003; main effect of group F = 16.1, p = 0.0003; Sidak’s multiple comparisons, OP-CH vs. OP-JF, p < 0.0001). GluA2 surface expression was not altered by JF in either group compared with controls  = 0.22, p = 0.6; CH vs. JF: F = 0.01, p = 0.9; OP vs. OR: F = 1.4, p = 0.2; GluA2: two-way ANOVA no group × diet interaction: F = 3, p = 0.1; CH vs. JF: F = 0.5, p = 0.5; OP vs. OR: F = 0.1, p = 0.7). This pattern is consistent with an increase in surface expression of CP-AMPARs and is consistent with previous results [We first determined the effects of 10 days of JF diet followed by 2 weeks of withdrawal on GluA1 and GluA2 surface expression in obesity-prone and obesity-resistant male rats . We found increases in GluA1 surface expression in both JF no deprivation and 24-h deprivation groups compared with controls  = 6.4, p = 0.006; Sidak’s multiple comparison CH vs. JF no deprivation, p = 0.005, CH vs. JF-dep, p = 0.02). Thus, a period of JF deprivation is not needed for increased GluA1 surface expression in obesity-prone males. In addition, JF did not affect GluA2 surface expression in either group . For these studies only obesity-prone rats were used, as we have yet to see effects of diet manipulation on AMPAR protein expression or synaptic transmission in obesity-resistant rats  = 19.5, p = 0.02; Sidak’s multiple comparisons: baseline vs. chow, p < 0.001, baseline vs. JF no deprivation, p = 0.0004, baseline vs. JF dep, p < 0.0001). However, Naspm produced a significantly larger reduction in eEPSC amplitude in the JF 24-h deprivation group than either control or JF no deprivation groups  = 4.4, p = 0.02; Sidak’s multiple comparisons: chow vs. JF no deprivation, p = 0.99; chow vs. JF dep, p = 0.04; JF no deprivation vs. JF dep, p = 0.05). Furthermore, the magnitude of Naspm sensitivity was comparable in chow controls and the JF no deprivation group . Thus, although surface CP-AMPAR protein expression is increased in both JF exposed groups, a period of JF deprivation is necessary for increased CP-AMPAR synaptic transmission.Next, we used whole-cell patch clamping approaches to determine the contribution of CP-AMPARs to synaptic transmission in NAc core MSNs following 10 days of JF with and without 24-h JF deprivation . Thn = 14 cells, six rats; JF no deprivation: n = 11 cells, five rats; JF 24-h deprivation: n = five cells, four rats). There was a significant increase in the percentage of silent synapses following 10 days of JF without deprivation compared with controls  = 13, p = 0.0001, Sidak’s multiple comparisons: chow vs. JF no deprivation, p < 0.05). Furthermore, the percentage of silent synapses following 24-h of JF deprivation was similar to that of chow fed controls . Taken with data above, results show that JF increases the number of silent synapses and suggest that the insertion of CP-AMPARs following JF deprivation may then lead to a reduction in silent synapses.The insertion of CP-AMPARs has been associated with the generation of silent synapses in the NAc shell following cocaine , 38. Then = 8) and obesity-resistant (n = 9) male controls. Interestingly, GluA1 surface expression was lower in obesity-prone vs. obesity-resistant rats maintained on chow  = 2.44, p = 0.03), while GluA2 surface expression was similar between these groups . This may suggest that basal levels of CP-AMPARs are lower in obesity-prone rats, allowing for greater increases following JF diet.In order to provide clues as to why junk-food produces glutamatergic plasticity in obesity-prone, but not obesity-resistant males, we conducted additional analyses comparing GluA1 and GluA2 expression and excitatory transmission between obesity-prone  vs. obesity-resistant rats  = 2.67, p = 0.01), indicative of lower levels of excitatory synaptic transmission in obesity-prone groups. Thus, lower basal levels of excitatory transmission in obesity-prone rats may enhance their capacity for subsequent diet-induced plasticity (see “Discussion”). Furthermore, the distribution of raw AMPAR or NMDAR amplitudes was similar across cells from obesity-prone and obesity-resistant rats  and obesity-resistant (n = 17) female rats. For these studies we focused on the 2-week deprivation time point because we first wanted to determine if there are persistent effects of JF in females, and because this is when effects had been established in males [Effects of JF diet on AMPAR subunit expression and excitatory synaptic transmission were also examined in obesity-prone  = 1.4, p = 0.2; CH vs. JF: F = 0.4, p = 0.5; OP vs. OR: F = 2.2, p = 0.2; GluA2 Surface: two-way ANOVA no diet × group interaction: F = 1.3, p = 0.3; CH vs. JF: F = 0.5, p = 0.5; OP vs. OR: F = 0.1, p = 0.8). Given that effects on surface protein expression and synaptic transmission are not always parallel , we also determined the contribution of CP-AMPARs to NAc core synaptic transmission following 2 weeks of JF deprivation in obesity-prone females . Consistent with biochemical data, JF did not alter Naspm sensitivity in obesity-prone females  = 0.5, p = 0.6). To determine effects on excitatory transmission more generally, we also measured the AMPA/NMDA ratio in MSNs from obesity-prone females . We found that an increase in the AMPA/NMDA ratio in JF fed vs. chow fed controls  = 2.32, p = 0.04). However, this effect may be driven by decreases in NMDAR-mediated eEPSC amplitude . Thus, results in females differed dramatically from those in males, with no evidence of JF altering AMPAR-mediated transmission (CP-AMPARs or non-CP-AMPARs) or AMPAR subunit protein expression.In addition, there were no differences in surface or intracellular GluA1 or GluA2 protein expression between obesity-prone and obesity-resistant females fed a chow diet . Specifically, the magnitude of increased GluA1 surface expression was similar when tissue was collected after 1 day of JF deprivation or no deprivation period . While we cannot rule out potential contributions of weight gain to effects observed here, results to date do not support strong relationships between weight gain and CP-AMPAR increases. Specifically, we find comparable CP-AMPAR increases (both expression and function) whether obesity-prone males are maintained on JF for 10 days or 30 days, and have found that even when food intake and weight gain are the same in obesity-prone vs. obesity-resistant rats CP-AMPAR transmission is increased in obesity-prone but not obesity-resistant males . FurtherWhen the effects of JF on NAc core synaptic transmission were examined in males, we found an increase in the contribution of CP-AMPARs after 1 day of JF deprivation compared with chow fed controls Fig. , replicaOne notable difference between food vs. cocaine effects is that increases in NAc CP-AMPAR transmission (core) following prolonged cocaine self-administration occur gradually during withdrawal; they are absent 1 day after the last cocaine self-administration session, begin to appear ~14 days later and are markedly increased by ~25 days , we also determined the proportion of silent synapses in obesity-prone males following JF consumption. There was a marked increase in the proportion of silent synapses following 10 days of JF consumption compared with chow fed controls Fig. . FurtherIncreases in silent synapses can be due to the removal of AMPARs from existing synapses, or to the addition of new synapses lacking AMPARs , 37, 50.Although we favor the interpretation that the reduction in silent synapses following JF deprivation is due to maturation of silent synapses because of CP-AMPAR insertion, similarly to cocaine withdrawal (above), there are caveats to the interpretation. First, it is not possible to determine which synapses CP-AMPARs are added to . SeWhen effects of JF were examined in females, we found no evidence for alterations in AMPAR expression or function. We began with biochemical studies in which we varied the duration of JF exposure, collecting NAc tissue  after 10 days or 1 month of JF access and 2 weeks of deprivation. There were no differences in surface or intracellular GluA1 or GluA2 expression in any group. It is possible that JF could have produced transient effects that returned to levels comparable with that of chow controls within the 2-week deprivation period. However, we chose this time point in order to identify effects that persist for reasonably long periods following JF removal, and that correspond to the timing of effects found in males.Because we did not attempt to control for the cycle in biochemical studies, and ovarian hormones can affect NAc glutamatergic transmission , it is pTo our knowledge this is the first examination of diet-induced NAc glutamatergic plasticity in females, and studies of CP-AMPARs following cocaine exposure have yet to be completed in females. In addition, there is surprisingly little information regarding NAc glutamatergic plasticity in females. However, one recent study found that estradiol applied to slices can alter NAc core mEPSC frequency and amplitude , and anoWe found that JF consumption increases CP-AMPAR surface expression and generates silent synapses in the NAc of male rats. In addition, a brief period of JF deprivation is needed for the synaptic insertion of CP-AMPARs and the maturation of silent synapses in males. In contrast, JF did not induce AMPAR plasticity in females but may instead alter NMDAR-mediated transmission. Thus, these studies reveal sex differences in the effects of JF on NAc synaptic plasticity. In addition, they provide novel insights into how essential food rewards alter NAc function.This work was supported by NIDDK R01DK106188 and NIDDK 1R01DK115526-01 to CRF; TEB was supported by NIDA R01DA040965; YA-C was supported by R01DK106188-02-S1 and 1F99NS108549-01; TLF was supported by NIDA T32DA007268 and R01DK106188; and ETJ was supported by NIDA R01DA040965. The authors declare no competing interests.Supplemental Tables CaptionSupplemental Table 1Supplemental Table 2


To investigate the protective effect of parecoxib against lungischemia-reperfusion injury (LIRI) in rats and the mechanism.Thirty rats were divided into sham-operated, LIRI and LIRI+parecoxib groups.LIRI model  wasconstructed in LIRI and LIRI+parecoxib groups. In LIRI+parecoxib group, 10mg/kg parecoxib was given via femoral vein 15 min before ischemia beginning.At the end of the reperfusion, blood gas analysis, lung wet to dry massratio measurement, lung tissue biochemical determination and hemeoxygenase-1 (HO-1) protein expression determination were performed.Compared with LIRI group, in LIRI+parecoxib group the oxygenation index wassignificantly increased, the alveolar-arterial oxygen partial pressuredifference was significantly decreased, the lung wet to dry mass ratio wassignificantly decreased, the lung tissue malondialdehyde content wassignificantly decreased, the lung tissue superoxide dismutase andmyeloperoxidase activities were significantly increased, the lung tissuetumor necrosis factor α and interleukin 1β levels were significantlydecreased, and the lung tissue HO-1 protein expression level wassignificantly increased .Parecoxib pretreatment can mitigate the LIRI in rats by reducing oxidativestress, inhibiting inflammatory response and up-regulating HO-1 expressionin lung tissue. Lung ischemia-reperfusion injury (LIRI) often occurs in lung transplantation,cardiopulmonary bypass, pulmonary embolism thrombectomy, isolated pulmonaryperfusion and other surgeriesCyclooxygenase (COX) is an important rate-limiting enzyme in the synthesis ofprostaglandins. COX has two isozymes, including COX-1 and COX-2. COX-2 is animmediate early gene, which is highly expressed induced by many factors. It ismainly located in the nuclear membrane. The prostaglandins produced by COX-2catalysis can preferentially enter the nucleus and regulate the transcription oftarget genesParecoxib is a specific COX-2 inhibitor commonly used in clinics. It has been foundthat parecoxib can attenuate the hepatic ischemia-reperfusion injury in rats byinhibiting the inflammation and oxidative stressIn the present study, the protective effect of parecoxib against LIRI in rats wasinvestigated, and the mechanism related to the oxidative stress, inflammation, andHO-1 expression were explored.Guide for the Care and Use of Laboratory Animals, by theNational Institutes of Health.This study was approved by the ethics committee of Zhejiang Hospital. All animalprocedures followed the Principles of Laboratory Animal Care and were in accordancewith the Male specific-pathogen-free sprague dawley (SD) rats (260-280 g) were used forexperiment. Before the experiment, the rats were fasted for 8 h and drankfreely. The anesthesia was performed by intraperitoneal injection of 1%pentobarbital sodium, with dose of 40 mg/kg. The rats were fixed in supineposition. A median neck incision was made to separate the trachea. The tracheawas cut open. The endotracheal tube was inserted into the trachea, and wasconnected to the small animal ventilator for mechanical ventilation. Theparameters of ventilator were as follows: tidal volume, 8 mL/kg; respiratoryrate, 60 times/min; ratio of inhalation to exhalation, 1:2. An incision was madein the right groin, and the femoral artery and femoral vein were separated. A24-gauge venous indwelling needle was placed. Then, the rats were transferred tothe right decubitus position. The thoracotomy was performed through the leftfifth intercostal space to separate the left hilum. A 100 U/kg heparin wasinjected through the femoral vein. After 5 min, the left pulmonary artery wasclamped with a noninvasive vessel clamp for 60 min of ischemia. Then, thevascular clamp was released, and the blood perfusion was restored for 120min.Thirty rats were randomly divided into sham-operated group, LIRI group andLIRI+parecoxib group, 10 individuals in each group. In the sham-operated group,only the left pulmonary hilum was separated by thoracotomy, without occlusion ofpulmonary artery. The complete LIRI model was established in LIRI group andLIRI+parecoxib group. In parecoxib group, at 15 min before pulmonary arteryocclusion, 10 mg/kg parecoxib was given via femoral vein. In the LIRI group andLIRI+parecoxib group, equal volume of normal saline was given via femoralvein.2) and partial pressure of carbondioxide (PaCO2) were recorded. The oxygenation index (OI) andalveolar-arterial oxygen partial pressure difference (PA-aO2) werecalculated as follows: OI = PaO2 / fraction of inspired oxygen(FiO2); PA-aO2 = (atmospheric pressure - saturatedwater vapor pressure) × FiO2 - PaCO2 / 0.8 -PaO2.After 120 min of reperfusion, the blood sample was collected through the femoralartery. The blood gas indexes were detected by automatic blood gas analyzer. Thepartial pressure of oxygen  content, superoxidedismutase (SOD) activity and myeloperoxidase (MPO) activity were determined byspectrophotometer. The tumor necrosis factor a (TNF-a) and interleukin 1ß(IL-1ß) levels were determined by enzyme linked immunosorbent assay. Thedetermination procedures were according to the instructions of the kits.The HO-1 protein expression in lung tissue was determined using western blottingassay. The lower one third of left lung tissue was taken and treated with 1-mLlysate. The total protein was extracted, and quantified by bicinchoninic acidmethod. A 50-µg crude protein sample was loaded on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The separated protein sample wastransferred to nitrocellulose membranes, followed by blocking with 5% bovineserum albumin. The membranes were incubated with rabbit anti-rat HO-1 polyclonalantibody and rabbit anti-rat ß-actin polyclonal antibody at 4°C overnight,respectively. Then, the membranes were incubated with horseradishperoxidase-labeled IgG at room temperature for 2 h. Finally, the membranes wereincubated with chemiluminescence reagent, followed by developing and scanning.The gray value of stripes on the membranes was measured using the gel imageanalysis system. The images were analyzed using ImageJ software. The ratio ofgray value of HO-1 protein stripe to that of ß-actin stripe presented theexpression level of HO-1 protein.All data were presented as the mean±SD. The statistical analysis was performedusing Statistical Package for the Social Sciences (SPSS) software. Thedifferences among three groups were assessed by one-way analysis of variancefollowed by post-hoc least significant difference (LSD)-t test. P < 0.05 wasconsidered to indicate a statistically significant difference.2 was significantlyhigher (P < 0.05). Compared with LIRI group, in LIRI+parecoxib group the OIwas significantly higher (P < 0.05), and the PA-aO2 wassignificantly lower (P < 0.05) , and the PA-aO < 0.05) .After 120 min of reperfusion, in sham-operated, LIRI and LIRI+parecoxib groups,the lung wet to dry mass ratio was 3.82±0.48, 5.79±0.92 and 4.55±0.63,respectively. The lung wet to dry mass ratio in LIRI and LIRI+parecoxib groupswas higher than that in sham-operated group (P < 0.05), but in LIRI+parecoxibgroup it was lower than in LIRI group (P < 0.05) .As shown in At the end of reperfusion, the lung tissue TNF-a and IL-1ß levels in LIRI andLIRI+parecoxib groups were significantly higher than those in sham-operatedgroups, respectively (P < 0.05). Compared with LIRI group, each index inLIRI+parecoxib group was significantly decreased (P < 0.05) .After 120 min of reperfusion, the western blotting assay showed that the lungtissue HO-1/ß-actin ratio in sham-operated, LIRI and LIRI+parecoxib groups was3.82±0.48, 5.79±0.92 and 4.55±0.63, respectively. The HO-1/ß-actin ratio in LIRIand LIRI+parecoxib groups was higher than that in sham-operated group (P <0.05), and that in LIRI+parecoxib group was higher than that in LIRI group (P< 0.05) .2 can be used as indicators tojudge whether the lung ventilation function is normal. It is found that parecoxibcan attenuate the hepatic, myocardial and cerebral ischemia-reperfusion injuryAfter LIRI, the alveolar capillary barrier is destroyed, the permeability of lungtissue is increased, and the exudation of plasma components was increased. It causesthe pulmonary interstitial edema and bleeding, leading to the ventilationdysfunction. The lung wet to dry mass ratio can indirectly reflect the degree ofpulmonary interstitial edema. OI and PA-aO2 was significantly increased, and the lung wet todry mass ratio was significantly increased. This suggests that there are pulmonaryinterstitial edema and lung ventilation function abnormality in LIRI rats. Comparedwith LIRI group, above indexes in LIRI+parecoxib group were significantly improved.It indicates that the parecoxib pretreatment can reduce the pulmonary interstitialedema and enhance the lung ventilation function in LIRI rats.In the present study, the protective effect of parecoxib against LIRI in rats wasinvestigated. Results showed that, after 120 min of reperfusion, compared withsham-operated group, in LIRI and LIRI+parecoxib groups the OI was significantlydecreased, the PA-aOThe production of free radicals is an important factor of ischemia-reperfusioninjuryIt is found that parecoxib can resist the oxidative stress in bodyExcessive inflammatory response plays an important role in the pathological processof LIRI. Many cells participate in the process of LIRI, leading to inflammatory cellinfiltration and release of a large number of cytokines such as TNF-a, IL-1ß andothersPrevious studiesHO-1 is a rate-limiting enzyme of heme metabolism in mammalian cells and can catalyzethe degradation of free heme to iron, carbon monoxide and biliverdin and reduce theproduction of oxygen free radicals by degrading free heme in damaged cellsThe parecoxib pretreatment can reduce the pulmonary interstitial edema and enhancethe lung ventilation function in LIRI rats. The mechanism may be related to itsreducing oxidative stress, inhibiting inflammatory response and up-regulating HO-1expression in lung tissue.This study still has some limitations. Firstly, only one dose of parecoxib wasinvestigated, and the dose-effect relationship is relatively simple. Secondly, thisstudy has not investigated other action mechanism of parecoxib. In next studies, thedose-effect relationship of parecoxib and the other action mechanisms of parecoxibon LIRI should be further explored.


Aspergillus puulaauensis strain MK2 was isolated from a dead hard tick . Here, we determined the chromosome-level genome sequence of A. puulaauensis MK2. Haemaphysalis longicornis hard tick study, we observed that the ticks were covered with fungi, resulting in subsequent tick death. H. longicornis is known as a vector of several pathogens causing illnesses, including Theileria spp., Babesia spp., Rickettsia spp., and severe fever with thrombocytopenia syndrome virus , pre-rRNA processing protein (tsr1), and RNA polymerase II subunit (rpb2) for strain MK2 and Aspergillus versicolor clade strains , and then the retrieved mycelia were subjected to DNA extraction using DNAs-ici!-F  and RNA extraction using RNAiso Plus  and the SV total RNA isolation system . The genomic DNA of strain MK2 was sequenced to 170- and 164-fold coverage using Oxford Nanopore Technologies (ONT) MinION and Illumina NovaSeq 6000 platforms, respectively, with PCR-free workflows. ONT and Illumina sequencing libraries were prepared using the ligation 1D kit SQK-LSK109; ONT) and the NEBNext Ultra II DNA library preparation kit , respectively. The ONT reads and Illumina reads were used for anu v2.0  with ONTanu v2.0  with Ill; ONT andanu v2.0 , AUGUSTUanu v2.0 , Glimmeranu v2.0 , and Genanu v2.0  via the y v2.8.5  and analy v2.8.5 , MIBiG vy v2.8.5 , Pfam v3y v2.8.5 , and dbCy v2.8.5   under accession numbers DRX256207, DRX251723, and DRX251722.The nucleotide sequences of


Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated. In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100 mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30 nm and suitable homogeneity with minimum 12-month stability at 4 °C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 h in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents’ delivery. Viruses have naturally evolved optimal and efficient targeted cargo delivery and, hence, have successfully been used by biologists for the same purposes. However, nearly all of the naturally occurring viruses that may be most suitable for a specific cargo delivery purpose are also pathogenic and/or posing other threats. Therefore, various modifications are applied to a virus before it could safely be used as a vector. Virus-like particles (VLPs), which are frequently used by biologists for targeted and efficient cargo delivery, are viral particles normally devoid of considerable pathogenicity4. The MS2 VLP is a 27 nm spherical RNA bacteriophage virus-like particle, consisting of 180 identical 129-amino acid coat protein subunits of ~ 14 kDa that form the icosahedral face for a T = 3 surface lattice7. MS2 VLP due to its small size and special shape, as well as its ability to encapsulate different nucleic acids, proteins, and other small molecules, and its endocytic route of entry has been considered a suitable vector for delivering into and tracking cargoes in the targeted cells8. The coat proteins of MS2 VLP can be recombinantly expressed and assembled in bacteria and yeasts12. It is conveniently prepared, packaged, delivered, and has high safety margins. Thus, the MS2 VLP has been used as a potential medicinal carrier13 for the treatment of various diseases such as prostate cancer14, hepatocellular carcinoma16, and other diseases20. It has also been used for various research purposes including the investigation of RNA trafficking in live cells21, in platforms of vaccines and peptide epitope identification27, and quality control of DNA and RNA virus detection30.Delivering small organic or inorganic molecules into the biological systems for therapeutic and research purposes has always been a challenge for biologists. In addition to natural barriers challenging the reach of the target organ and further, inside the cells, the stability of the material of interest to be delivered and that of the delivering agent, plus their safety and toxicity levels introduce additional limitations and challenges. In this line, various approaches such as those involving nanoscale platforms have been tested to find optimal delivery agentspac site), which in turn binds to coat protein dimers33. Thus, the interaction with the 5’-terminus of the so-called pac site has been used as a preferred strategy to encapsulate different cargoes8. In this respect, it is very important to understand the behavior and assembly of virus-like particles and their aggregation and stability in different conditions. To make efficient VLPs, various physicochemical factors such as pH, temperature, and ionic strength of the solution, the ideal size, homogeneity, and stability of produced VLPs, and their capability to protect cargoes from degradation need to be taken into account. Likewise, in the recombinant production of MS2 VLPs, upon the transformation of plasmids expressing MS2 coat protein and a part of maturase into the bacteria, the most appropriate pH and buffers to support the stability and homogeneity of VLPs are sought after36.It is well characterized that nucleic acids and other small molecules can be packaged in the MS2 VLP via their interaction with a short RNA stem-loop structure  of NaNO3 buffer were merely seen at a pH value near the isoelectric pH (between 3 and 4)9. In this study, we introduce a new and optimized buffer to prepare the most stable condition for VLPs. To simulate the in vivo conditions, we assess the stability of VLPs produced in this buffer after incubation in 50% FBS and at 37 °C temperature. We report improved buffer conditions that are used for the preparation of VLPs with appropriate size, shape, and stability.Different studies have shown that the amounts of electrolytes are important factors for the assembly of MS2 bacteriophage and related VLPsTo confirm the expression of the coat protein of VLPs, ~ 14 kDa disassembled protein of purified MS2 VLPs was separated by electrophoresis on 12.5% acrylamide gel and detected using specific antibody and Western blot method. BL21 (DE3) bacterial lysate showed both monomers and dimers of coat protein  and pH values (7 and 8) were assessed in MS2 VLP expression, production, and purification by the PEG precipitation method. The reduction of NaNO3 concentration could increase the particle size to larger than 100 nm . VLPs in NaNO3-PBS (pH:7.4) displayed a much smaller size than the native MS2 bacteriophage (27-30 nm). PDI measurement of VLPs produced in NaNO3-Tris (pH:7.4) showed a wide size distribution in comparison to VLPs in NaNO3-Tris (pH: 8), which was also confirmed by TEM analysis  in Tris , HEPES (pH: 7.4 and 8), and PBS (pH:7.4) were examined. In general, NaNO3 addition to various buffers boosted the VLP preparation efficiency. Although the particles in NaNO3-HEPES (pH:7.4) were smaller than particles in NaNO3-HEPES (pH:8) , yet the size of particles in NaNO3-HEPES (pH:7.4) was bigger than the size of the native MS2 bacteriophage (27–30 nm)  of MS2 VLPs were negative in all solutions and buffers except for the VLPs in PBS (pH:7.4). Results were shown the variable electrophoretic mobility for VLPs in PBS alone or with 100 mM NaNO3   solution and NaNO3-Tris  buffer, protein concentration was measured by spectrophotometry. Protein concentration and the number of VLPs in NaNO3 (pH:8) solution decreased over time , while it was stable for VLPs in NaNO3-Tris (pH:8) (≈40 mg ml15) Fig. a.Figure Considering the RNA sequence packed in MS2 VLPs Fig. e, we per39. Although the PDI values in all three experiments were below 0.45 and, in the midrange, the effect of different concentrations of serum on PDI variation was evident in NaNO3  solutions  solutions and NaNO3-Tris  buffer were incubated with different serum concentrations . While VLPs in NaNO3  had a bigger size than VLPs in the same buffer with pH:8, by rising serum concentrations, a decreasing trend of VLP size was observed which was an indicator of serum effects on PEG dispersity. In contrast, the size of VLPs prepared in NaNO3-Tris  buffer was stable in different serum concentrations Fig. a, b, c. RNA extraction and RT-PCR experiments for VLP samples generated in NaNO3-Tris buffer (pH:8) in the presence of 50% serum concentration revealed the existence of RNA sequences and their stability up to 48 h at 37 °C  which could not transfect easily. Immunocytochemistry and flow cytometry was then performed using the MS2 bacteriophage coat protein antibody. Bright intense red spots seen after 1 h, were then dispersed in the cytoplasm after 4 h, indicating the VLP entrance and probable disassembly of the VLPs over the time . This buffer greatly enhanced the consistency  precipitation44, is the less-toxic and low-cost method, appropriate for purifying proteins from various protein sources45. In addition, the presence of this uncharged polymeric layer on the outer periphery of the particles leads to an electrostatic and hydrodynamic screening of the negatively-charged inner RNA content of MS2 VLPs, which reduces the effect of internal constituents on the electrophoretic mobility of the particles2. Previous studies have shown that various physicochemical factors can affect the capsid stability and aggregation of VLPs33. Among these, pH and ionic strength are the most important factors. Na+ containing solutions are used as the most acceptable ionic solutions in the PEG precipitation method46, considering the fact that in monovalent electrolytes such as NaCl, KCl, and LiCl, aggregation of MS2 does not occur within a reasonable kinetic time frame, and MS2 was stable even at high salt concentrations35. Thus, we used the NaNO3 solution to prepare an appropriate ionic environment. The shape of VLPs was analyzed by the TEM method. Furthermore, the particle size and PDI were measured as pharmaceutical characteristics of particles that influence endocytosis and cellular uptake.Along with VLP synthesis, various purification strategies such as dialysis2, pH effectiveness2, and minimum mobility46. Despite the previous studies showing the stability of virus nanoparticle suspension against aggregation in ≤ 1 M NaCl for more than 10 h35, in this study, the same assessment of two concentrations at the stable pH:8 resulted in different sizes of VLPs  decreases with increasing salt concentration as a result of the screening of the virus charge by the ions present in the electrolytic medium, tending to non-zero plateau value for solutions ionic strengths above 100 mM. This latter further emphasizes the better properties of using 100 mM NaNO3 to be used for VLP purification. This negative charge of the particles on the other hand, minimizes the likelihood of aggregation as a result of significant repulsive electrostatic interactions53.Irrespective of the purification method, the preparation of MS2 VLPs in two concentrations of NaNO3 solution (1 mM and 100 mM) in different ranges of pH (2 to 7) revealed higher stability of VLPs in 100 mM NaNO3 (pH:7) because of its high electrolyte concentration9. VLPs produced in 100 mM NaNO3 at pH,8 displayed a smaller size (p < 0.0001) compared to VLPs produced at pH,7  are more uniform and smaller than VLPs in HEPES and PBS buffers , HEPES pH:7.2–7.4), and PBS (pH:7.4) buffers, alone or mixed with some electrolytes and chemicals such as MgSO4, NaCl, and EDTA in different molarities have been chosen in various protocols as a protective buffer for VLP preparation and storage–7.4, and35 and to improve the VLPs’ synthesis process, we employed the new VLP formation strategy by mixing Tris, HEPES, and PBS buffers with NaNO3 . VLPs in Tris buffer  containing NaNO3 (100 mM) displayed a similar size to MS2 bacteriophage which is the convenient size for delivering, whereas VLPs in the same buffer at pH: 7.4 showed the maximum size among all the newly developed buffers  are stable for 3 months at 4 °C, preparation of VLPs in NaNO3-Tris buffer  can increase the stability period to more than 12 months  did not affect the size. Moreover, in the presence of 50% serum at 37 °C, the shRNA sequence inside the VLPs was protected against degradation for 48 h , and also the reliable cell entry properties Fig. , represeNo animal was used in this study.18. The amplified sequence was inserted in-frame into pACYCDuet plasmid  through BamH1 and HindIII restriction enzyme  sites. The oligonucleotide sequence encoding the shRNA sequence was designed by siRNA Wizard ™software   was sub-cloned into the other multiple cloning sites of pACYCDuet plasmid through BglII and Kpn1 restriction enzyme  sites  Table 18. The ates Fig. e. This sE. coli Bacteria as a prokaryotic expression system. The Ecoli BL21(DE3)-SC Duet was cultured in a terrific broth medium at 37 °C, supplemented with 34 µg/ml chloramphenicol (pACYCDuet antibiotic selection). The expression of the sequences was induced by 1 mM IPTG  at an OD 600 = 0.6 for 16 h at 22 °C. Bacteria were precipitated by centrifugation , and the derived precipitate was resuspended in the appropriate buffer . Lysozyme treatment (0.05 mg ml-1) was performed for 30 min at room temperature followed by sonication . Cell debris was collected by centrifugation . The supernatant containing VLPs was filtered through a 0.22 µm membrane  and stored at 4 °C for further examination2. To eliminate cell debris, the suspension was filtered through a 0.1 µm membrane  and then MS2 VLPs were purified according to the polyethylene glycol (PEG) precipitation method44. In brief, the precipitation of VLPs was performed by adding PEG  at 10% of the final concentration (w/v) and 500 mM NaCl. The suspension was incubated at 4 °C for 18 h and subsequently was centrifuged at 10000 g for 1 h, 4 °C. The pellet containing MS2 VLPs was resuspended in the buffer used in the first step of purification . Eventually, this MS2 VLP suspension was filtered through a 0.22 µm membrane . All MS2 VLP samples were kept at 4 °C. Solutions of 1 mM, and 100 mM NaNO3 , 1 M Tris  buffer, and HEPES Buffer  were prepared fresh, one day before experiments.The SC Duet plasmid was introduced into the BL21(DE3) strain of g, 20 min). Then, we assessed the expression of coat protein by SDS-PAGE (12.5% acrylamide gel). Proteins were transferred to a 0.45 µm nitrocellulose membrane . The membrane was blocked with 5% non-fat dry milk overnight at 4 °C and then incubated with a polyclonal Anti-Enterobacteria Phage MS2 coat protein antibody  for 3 h at room temperature. Incubation with HRP-labeled secondary antibody  for 1 h was performed to confirm the presence of 14 and 28 k Da bands. The membrane was developed with Pierce™ ECL Western Blotting Substrate . Signals were detected using the Chemiluminescence Detector system .To confirm the expression of VLP coat proteins, purified MS2 VLPs were disassembled by diluting 1:2 in 20 mM cold glacial acetic acid for 1 h at 4 °C. To precipitate the free nucleic acids, samples were centrifugated at 4 °C  at 120 kV at 4000×, 10,000×, 25,000×, 31,500×, 50,000×, 63,000×, and 80,000× magnifications.MS2 VLPs were diluted 1:4 in selected buffers and solutions. Particle size and polydispersity index (PDI) were measured by particle size analyzer  based on Dynamic Light Scattering (DLS), 657 nm at 25 °C. Samples were analyzed by cumulants analysis of 5 consecutive measurements.Zeta potential for those samples which were not stored in Buffer was estimated by Zeta potential detector  and reported as zeta potential (mV) line plot at 25 °C. To ensure reliability, it has been performed at least three times.µ) of MS2 VLPs was measured at 23 ± 1 °C using Zeta compact-CAD (France) as a function of buffer containing different electrolytes at pH 7 and 8 for PEG-precipitated VLPs after filtering via a 0.1 µm membrane to remove all the aggregated particles from the suspension. VLPs were diluted at 1:10 in selected buffers and solutions. Experiments were carried out at least three times.Electrophoretic mobility , one day before the experiment. Evaluations were done in triplicates at 0 h, 3 h, 12 h, 24 h, 7 days, and 1-month time points.280)-1 Abs in 1 cm (1 mg ml−1) . Measurements were performed three times at different time points . According to the protein concentration and the molecular mass of each VLP particle (14 kDa per coat protein × 180 coat proteins per VLP = 2520 kDa), the number of VLPs in 100 mM NaNO3 solution and NaNO3-Tris buffer (pH:8) was calculated:Protein concentration was measured by absorbance at 280 nm (AEach VLP molecular mass = 2520 kDa = 4.18e−15 mg.−1)/4.18e−15.Number of VLPs (in each milliliter) = protein concentration of VLP suspension  (1:1), incubated in 37 °C for 2, 4, 6, 8, 10, 12, 24, and 48 h, and then stored in − 80 °C for further RNA isolation, reverse transcription, and PCR. Furthermore, MS2 VLPs were also incubated in different serum concentrations  for 1 h at 4 °C and particle size and PDI were evaluated diluting 1:4 with additional buffer.300 ul of MS2 VLP suspension in different solutions or buffers (after purification by PEG precipitation method) were subjected to RNA isolation using the Total RNA Isolation Kit  according to the manufacturer’s instructions. The quantity of RNA was determined by a spectrophotometer . A total of 1 µg RNA was used for cDNA synthesis using random hexamer and MMLV reverse transcriptase . To identify and check the integrity of shRNA-4pac RNA, polymerase chain reactions were carried out by Taq DNA Polymerase Master Mix  and designed primers  were cultured in Dulbecco’s modified minimal essential medium   containing 10% (v/v) of FBS  and 1% penicillin/streptomycin  at 37 °C, 5% CO2 and 95% humidity. Cells were grown on poly D-lysine  coated coverslips in four groups , and blank negative control (with VLP treatment and without primary antibody). Briefly, cells were treated with 0.05 µg/ml of VLP suspension 24 h after seeding. The cells were fixed by 2% paraformaldehyde (w/v) for 10 min at room temperature. Subsequently, after washing with cold PBS, the cells were permeabilized with 0.25% Triton X-100 in PBS) for 10 min at room temperature and blocked with PBS containing 1% BSA. Cells were then incubated with primary antibody  for 3 h at room temperature. The coverslips were washed with cold PBS and then incubated for 1 h at room temperature in darkness with a secondary antibody . The coverslips were mounted on slides after washing with cold PBS. Stained cells were visualized using fluorescent microscopy .The quantitative analysis of VLP entrance to Neuro2A cells was performed by flow cytometry. Cells were cultured as previously described in the immunocytochemistry section. Neuro2A cells were treated with 0.05 µg/ml of VLP suspension 24 h after seeding and prepared for flow cytometry in four groups , and blank negative control (with VLP treatment and without primary antibody). Following the cell incubation with VLP (1 and 4 h) and washing with PBS to remove additional VLPs, the cells were trypsinized and subjected to flow cytometry. Followed by Fixing with 4% paraformaldehyde (w/v) for 30 min at room temperature, permeabilizing by 0.2% Triton X-100 (15 min), and blocking with 0.5% BSA and 2% FBS for 30 min on ice. Subsequently, the cells were incubated with primary antibody solution  for 3 h at room temperature and then were incubated for 1 h at room temperature in darkness with a secondary antibody . Interval of all steps the cells were washed with 0.5% FBS-PBS  and were centrifuged at room temperature . the cells were diluted in PBS and analyzed with BD ™Accuri C6 Flow Cytometer (USA). Results were analyzed using FlowJo software  and were shown as an intensity plot . Results were analyzed using the Mann–Whitney and Kruskal Wallis test analysis.All charts of pictures were set and labeled in Adobe photoshop (2020) and were saved in TIFF format. TEM microscopic and cell images were used without any change. Imaging for ICC experiments was done by Nikon fluorescent microscope in the same condition for all shots at 10× magnification (NIS-Elements F 4.60.00 64-bit software). Agarose gels are full-length versions and just a little brightness and contrast were changed for the entire gels. A full-length original version of the blot was used. All graphs were designed with GraphPad Prism .Supplementary Figures.


TL1A gene. TL1A siRNA remarkably intervened EMT formation induced by TNF-a in the Beas-2B cells. In addition, EMT was induced by the addition of high concentrations of recombinant human sTL1A with the cell medium. The TL1A overexpression via pc-mTL1A in vitro remarkably increased the EMT formation induced by TNF-a. Overall, these findings indicate that the TL1A/DR3 axis may have a therapeutic role for asthmatic with high TNF-a level.Tumor necrosis factor (TNF)-like cytokine 1A (TL1A), a member of the TNF family, exists in the form of membrane-bound (mTL1A) and soluble protein (sTL1A). TL1A binding its only known functional receptor death domain receptor 3 (DR3) affects the transmission of various signals. This study first proposed that the TL1A/DR3 axis was significantly upregulated in patients and mice with both asthma and high TNF-a expression and in TNF-a-stimulated epithelial Beas-2B cells. Two independent approaches were used to demonstrate that the TL1A/DR3 axis of mice was strongly correlated with TNF-a in terms of exacerbating asthmatic epithelial–mesenchymal transformation (EMT). First, high expression levels of EMT proteins  and TL1A/DR3 axis were observed when mice airways were stimulated by recombinant mouse TNF-a protein. Moreover, EMT protein and TL1A/DR3 axis expression synchronously decreased after mice with OVA-induced asthma were treated with infliximab by neutralizing TNF-a activity. Furthermore, the OVA-induced EMT of asthmatic mice was remarkably improved upon the deletion of the TL1A/DR3 axis by knocking out the  Asthma, a heterogeneous disease, is characterized by chronic inflammation, epithelial–mesenchymal transformation (EMT), and airway remodeling . With thChronic airway inflammation disorder exacerbates the progression of asthma . After iTNFSF15) is a type II transmembrane protein with a stable trimer structure similar to TNF-a  and mouse TNF-a (AF-315-01A) were obtained from PeproTech. The pcDNA3.1 and pc-mTL1A were constructed from FengHuiShengWu Biotechnology. Human TL1A siRNA (sc-39846) was obtained from Santa Cruz Biotechnology. TNF-a (ab215188) and collagen I (ab260043) antibodies were obtained from Abcam. Infliximab (A2019) was provided by Selleck Technology. Recombinant human sTL1A (1319-TL) was obtained from RD Systems. Antibodies of a-SMA (#19245), vimentin (#5741), and N-cadherin (#13116) were obtained from Cell Signaling Technology. Fibronectin (ET1702-25), GAPDH (ET1601-4), and HRP conjugated goat anti-rabbit IgG (HA1001) were from HUABIO. DR3 (#PA5-19882) was obtained from Invitrogen Technology. TL1A antibody (DF3053) was constructed by Affinity Biosciences. The HE staining kit (Cat#G1120), Masson staining kit (Cat#G1340), and broad spectrum SP kit (SP0041) were producted from SolarBio.via bronchoscopy from patients diagnosed with asthma according to the 2019 Edition of the Global Asthma Initiative Criteria. The characteristics of human samples are shown in Healthy bronchial epithelial tissue samples were obtained from patients with suspected airway tumors, but no histopathological evidence of disease was found in these samples. Asthmatic samples were collected Beas-2B cells  were derived from human bronchi and had epithelial-like appearance. Beas-2B cells were cultured in 6- or 24-well petri dishes (DMEM containing 10% FBS) to a density of 50%–70% and then used for all tests.Beas-2B cells were stimulated in cell culture medium with TNF-a (50 ng/mL) for 12, 24, and 48 h to observe the effects on EMT and TL1A/DR3 axis. Then, we used 12.5, 25, and 50 ng/mL TNF-a for stimulation for 48 h in the Beas-2B cells.Different concentrations of recombinant human sTL1A protein in the Beas-2B cell culture were stimulated for 48 h to observe the effects of exogenous sTL1A on EMT. In addition, cells were assigned to the control, NC siRNA, TNF-a, and TL1A siRNA+TNF-a groups. Cells in the NC siRNA group were transfected with NC siRNA by Lipofectamine 3000 for the negative control. Cells in the TNF-a group were cultured in cell culture medium with TNF-a (50 ng/mL) stimulation 48 h. Cells in the TL1A siRNA+TNF-a group were transfected with 20 nM TL1A siRNA to intervene TL1A for 12 h, and then stimulated with 50 ng/mL TNF-a to observe the effect of TL1A intervention on TNF-a-induced EMT.TL1A overexpression was established by pc-mTL1A to observe the effects on the TNF-a-stimulated Beas-2B cells. Cells in the control group were harvested when the density reached 50%–70% without any treatment. Cells in the pcDNA3.1 group were transfected with pcDNA3.1 by using Lipofectamine 3000 for the negative control. Cells in the pc-mTL1A group were transfected with pc-mTL1A by using Lipofectamine 3000 to overexpress mTL1A. Cells in the TNF-a group were cultured in cell culture medium by stimulating with 50 ng/mL TNF-a for 48 h. Cells in the pc-mTL1A+TNF-a group were transfected with pc-mTL1A by using Lipofectamine 3000 according to the manufacturer’s protocol, and then cells in the cell culture medium were stimulated with 50 ng/mL TNF-a for 48 h.+/-TL1A female and male mice (18–20 g) were obtained from Nanjing University Institute of Zoology, and then bred and raised in the Qianfoshan Hospital at SPF environment. Female and male +/-TL1A mice were mated, and the pups of -/-TL1A and +/+TL1A females were selected via genetic identification. According to the international asthma animal model, female mice were selected for asthma study . The effects of TNF-a on the TL1A/DR3 axis and airway EMT in mice were assessed using two independent methods. First, the airways of wild female mice were stimulated with mouse TNF-a protein. Second, wild female mice were divided into the control, infliximab, OVA, and OVA+infliximab groups. The preparation of mouse model is illustrated in Wild female mice were randomly divided into the control and OVA groups to observe the changes in TNF-a and TL1A/DR3 axis  was added, oscillated at 600 rpm for 1 min, incubated at 37 °C for 16–18 h, and then subjected to enzymatic hydrolysis. After adding 0.1% TFA solution, the peptide was desalted using C18 cartridge and freeze-dried in vacuum. After enzymatic hydrolysis, the peptide was dried and redissolved with 0.1% TFA, and then desalted in the thermo desalting spin column to quantify the peptide. An appropriate amount of peptide was obtained from each sample for chromatographic separation by using an EASY-nLC 1200 chromatography system (Thermo Scientific). The obtained LC-MS/MS RAW files were finally imported into the search engine sequest HT in proteome discoverer software  for database retrieval. The above lung tissue samples were tested by Shanghai Bioprofile Technology Company Ltd.The control (N) group and OVA-induced asthma (M) group consisted of three and four mouse samples, respectively. Label-free 10-standard reagent was used for labeling and quantification. An appropriate amount of lung tissues was added with 200 µL of SDT lysate for ultrasonic crushing in ice bath. Lung supernatant was obtained after centrifugation at 4 °C and 16,000 ×g. Lung protein samples (20 µg) were obtained from each group at 5:1 (V/V), added into the 5× loading buffer solution, and then bathed in boiling water for 5 min. SDS-PAGE gel electrophoresis and coomassie blue staining were performed. Lung protein samples (300 µg) were obtained from each sample for enzymatic hydrolysis. A proper amount of 1 M DTT was added to each sample to a final concentration of 10 mM. Then, the sample was bathed in boiling water for 5 min and cooled to room temperature. An appropriate amount of 800 mM IAA was added to a final concentration of 50 mM. The solution was shaken at 600 rpm for 1 min and kept away from light for 30 min at room temperature. Add 6 times the volume of cold acetone reagent, and the solution was incubated overnight at −20 °C to precipitate the protein. The solution was centrifuged at 16,000 ×g for 30 min, the supernatant was removed, and the protein precipitate was retained. An appropriate amount of acetone reagent was added to clean the protein precipitate, and the process was repeated twice. The protein precipitated sample was dried in a fume hood to remove the organic reagent. Approximately 150 µL of trypsin buffer (6 µg trypsin in 144 µL NHThe whole-body plethysmography (WBP) system was located in a separate room to keep the surrounding environment quiet. The mice were placed in the test box and allowed to adapt to the environment for 30 min. The airway resistance indexes including Penh were measured using the WBP system with gradient concentrations of acetylcholine stimulation .Mice were added with 4% paraformaldehyde to fix the lung tissue through pulmonary circulation. The lung tissue was cut into blocks with dimensions of 0.3 cm×0.3 cm×0.5 cm, embedded in paraffin, and cut into 5 µm-thick sections. Hematoxylin-eosin staining (HE) was used to assess the degree of airway inflammation . CollageLung tissue sections were immersed in citric acid repair solution to perform antigenic repair under high temperature and pressure. After natural cooling, lung tissue sections were incubated with serum and antibody  for 12 h at 4 °C. The next day, the lung tissues were washed thrice with PBS (5 min/time), and the goat anti-rabbit antibody (1:200) was incubated in a dark room for 30 min. Finally, the lung tissue sections were stained with DAB and hematoxylin, followed by dehydration and transparency. Finally, the protein expression was evaluated by microscopy and Image J analysis.The concentration of different protein samples was detected using BCA protein assay kit. The protein samples in 10% separation gel were separated by electrophoresis at 80 V for 30 min and 120 V for 70 min, and then transferred to 0.45 or 0.2 µm PVDF membranes. Antibodies  were incubated at room temperature for 7 h after PVDF membrane infiltration with 3% BSA for 70 min. The PVDF membrane was washed with TBST solution, and the second antibody  was incubated for 1.5 h. Finally, TBST was used to clean the PVDF membranes, and chemiluminescence reagent was used to observe changes in PVDF membrane protein expression.Lung tissue sections were dewaxed, dehydrated, and treated with citric acid repair solution for antigenic thermal repair. After natural cooling and PBS washing, the lung tissue sections were sealed with goat serum for 30 min. The first antibody  was incubated on the surface of lung tissue sections for 12 h at 4 °C. Finally, the lung tissue sections were washed by PBS, incubated with fluorescence secondary antibody  and DAPI , and photographed under a fluorescence microscope.Slides were placed in the 24-well plate under aseptic conditions, in which Beas-2B cells were evenly planted. Beas-2B cells were treated with 4% paraformaldehyde for 30 min. After washing with PBS, Beas-2B cells were incubated with the first antibody  at 4 °C for at least 12 h. Beas-2B cells were washed with PBS, incubated with fluorescent secondary antibodies  and DAPI , and photographed under a fluorescence microscope.Data are presented as mean ± SEM. Two-tailed and unpaired t-tests were used for comparison between two groups. One-way ANOVA was performed for comparison among multiple groups. P < 0.05 was considered statistically significant.The Log2 scaled intensity was close to the same horizontal line, indicating good data quality and repeatability  was used to identify the protein levels of TL1A and TNF-a which were higher in normal lungs than in most other organs  in external cell culture  . ATF3 plTL1A knockout mice were constructed in vivo to explore the role of TL1A/DR3 in asthma. However, DR3 knockout mice has not been constructed because of financial and technical reasons.Although this study supports the conclusions to a certain extent, some limitations were observed. The higher concentration of sTL1A required to induce EMT might be due to various possibilities, and one of them is the much weaker bioactivity of commercial recombinant protein. In addition, In conclusion, this study is the first to demonstrate that TNF-a may regulate EMT in epithelial cells through the TL1A/DR3 signaling pathway. This study also provides a new theoretical basis and ideas for the treatment of asthma in the future.via the PRIDE partner repository with the dataset identified PXD031079. Further inquiries can be directed to the corresponding author.The original contributions presented in the study have been uploaded to the ProteomeXchange Consortium The studies involving human participants were reviewed and approved by Qianfoshan Hospital Institutional Review Committee. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Qianfoshan Hospital Institutional Review Committee. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.DZ conducted the study, performed part of the animal and cell operations, drafted the figures, and wrote the original draft. HY performed part animal operations, wrote and proofread the original draft. X-LD made some contributions to the manuscript revision work during the COVID-19 pandemic. J-TZ performed part of the animal operation and drafted some figures. JZ was provided human bronchial epithelial samples. W-JC interpreted the data and drafted the figures. YP, X-FL, and J-WX fed the Beas-2B cells, drafted some figures, and revised the manuscript. Z-HW provided big data support. LD conceived the idea, provided experimental funds, and performed rigorous revision. All authors approved the submitted version of the article.The present study work was supported by the National Natural Science Foundation of China (81770029), Key Research and Development Program of Shandong Province (2021SFGC0504), and Shandong Provincial Natural Science Foundation (ZR2021LSW015).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.


P = 0.044). These findings indicate that GULP1 represents a key downstream effector of AR signaling in enhancing sensitivity to cisplatin treatment.The underlying molecular mechanisms of resistance to cisplatin-based systemic chemotherapy in bladder cancer patients remain to be elucidated, while the link between androgen receptor (AR) activity and chemosensitivity in urothelial cancer has been implicated. Our DNA microarray analysis in control vs. AR knockdown bladder cancer lines identified GULP1 as a potential target of AR signaling. We herein determined the relationship between AR activity and GULP1 expression in bladder cancer cells and then assessed the functional role of GULP1 in cisplatin sensitivity. Androgen treatment in AR-positive cells or AR overexpression in AR-negative cells considerably reduced the levels of GULP1 expression. Chromatin immunoprecipitation further showed direct interaction of AR with the promoter region of GULP1. Meanwhile, GULP1 knockdown sublines were significantly more resistant to cisplatin treatment compared with respective controls. GULP1 knockdown also resulted in a significant decrease in apoptosis, as well as a significant increase in G2/M phases, when treated with cisplatin. In addition, GULP1 was immunoreactive in 74% of muscle-invasive bladder cancers from patients who had subsequently undergone neoadjuvant chemotherapy, including 53% of responders showing moderate (2+)/strong (3+) expression vs. 23% of non-responders showing 2+/3+ expression ( Urinary bladder cancer, mostly urothelial carcinoma, has been one of the most commonly diagnosed malignancies, especially in men ,2. MoreoAlthough new therapeutic options, such as immune checkpoint blockade, have become available, cisplatin (CDDP)-based combination chemotherapy remains the first-line standard for locally advanced or metastatic urothelial carcinoma ,6. HowevThe underlying mechanisms for CDDP resistance are still to be established, while it has been suggested that multiple molecular pathways, including extracellular signal-regulated kinases (ERKs) and PTEN, are involved in the modulation of the cytotoxic and anti-proliferative activity of CDDP ,10. MeanWe recently employed DNA microarray analysis in control AR-positive UMUC3 versus a UMUC3 subline stably expressing AR-shRNA . Of thosWe first examined the expression of GULP1 in four human bladder cancer lines that were known to be AR-negative  or AR-positive  . WesternA bioinformatics-driven search identified three potential AR binding sites in the GULP1 promoter region. We therefore investigated whether AR could regulate the expression of GULP1, using a chromatin immunoprecipitation (ChIP) assay . DNA fraNext, we assessed the impact of GULP1 expression on sensitivity to CDDP treatment in bladder cancer cells. To compare the cytotoxic effects of CDDP in GULP1-positive versus GULP1-negative cells, we silenced GULP1. As expected, the levels of GULP1 were substantially lower in 647V a and UMUUsing control-shRNA vs. GULP1-shRNA sublines, we assessed the role of GULP1 in the cell proliferation (via MTT assay a), apoptWe stained immunohistochemically for GULP1 in two separate sets of bladder tissue microarrays (TMAs). Positive signals were detected predominantly in the cytoplasm of non-neoplastic and neoplastic epithelial cells a.P = 0.396 (P = 0.285), as well as in those with muscle-invasive disease between GULP1-low and GULP1-high tumors (0 vs. 1+/2+/3+: P = 0.744 (P = 0.193).In the first set of TMA consisting of 129 cases of urothelial neoplasms and corresponding 89 non-neoplastic normal-appearing urothelial tissues, GULP1 was positive in 80 (90%) of non-neoplastic and 96 (74%) of neoplastic specimens . Thus, t = 0.396 b; 0/1+ v = 0.744 c; 0/1+ vP = 0.044).In another set of TMA consisting of muscle-invasive bladder cancer specimens from those who had subsequently undergone CDDP-based neoadjuvant chemotherapy, GULP1 was positive in 32 (74%) of 43 cases (1+: 17 (40%); 2+: 14 (33%); 3+: 1 (2%)). When GULP 0/1+ vs. 2+/3+ cases were compared, low expression was seen in 8 (47%) responders and in 20 (77%) non-responders . Thus, lP = 0.262).In our previous study , AR was Although resistance to CDDP-based chemotherapy is not uncommonly seen in patients with urothelial cancer, its underlying mechanisms are not fully understood. Meanwhile, AR activation in bladder cancer cells has been implicated to be associated with chemoresistance ,22,23,24GULP1 mRNA expression were found to be low (or undetectable) in all nine bladder tumors from patients who did not respond to CDDP therapy but were relatively high in two of six responders [Caenorhabditis elegans CED-6 and its human homologue GULP1, as adapter proteins, play an important role in phagocytosis ,25. Specsponders . It is asponders .n = 17) compared with non-responders (n = 26). We additionally demonstrated that the levels of GULP expression were significantly or marginally lower in urothelial neoplasms  and high-grade or muscle-invasive tumors (vs. lower grade or non-muscle-invasive tumors), while we failed to show a strong association between the expression level of GULP1 and the prognosis of the patients who had never received CDDP (prior to tumor recurrence or disease progression). These results indicate that GULP1 plays a critical role in increasing sensitivity to CDDP. Inconsistent with previous observations, however, we found no considerable impact of GULP1 on the proliferation/migration/invasion or apoptosis in 647V cells cultured in the absence of CDDP.Here, we confirmed some of the previous findings  in two oAs mentioned above, it remains unclear how AR signals modulate chemosensitivity. Based on our DNA microarray analysis data , we expeWhile AR activity must be considerably higher in men than in women, no studies have demonstrated significant sex-related differences in the prognosis of bladder cancer patients who undergo systemic chemotherapy. This may be due to a similar role of estrogen signaling in modulating chemosensitivity. Indeed, we recently showed an association between estrogen receptor (ER)-β activation and CDDP resistance in bladder cancer . AccordiAn inverse association between AR and GULP1 expression was seen in bladder cancer cell lines. In surgical specimens, we further demonstrated marginal or significant down-regulation of GULP1 expression in high-grade or muscle-invasive tumors. The levels of AR expression should therefore be elevated in high-grade/muscle-invasive bladder cancers compared with low-grade/non-muscle-invasive tumors. However, conflicting data exist regarding the expression of AR mRNA ,37 and pWe obtained anti-AR (N-20), anti-GULP1 (E-4), and anti-GAPDH (6c5) antibodies from Santa Cruz Biotechnology . DHT, HF, and CDDP were from Sigma-Aldrich .®® shRNA Bacterial Stock, Millipore Sigma, Burlington, MA, USA) was stably expressed in 647V and UMUC3 lines. These parental lines and sublines were maintained in DMEM  supplemented with 10% fetal bovine serum (FBS), penicillin (50 U/mL), and streptomycin (50 μg/mL) at 37 °C in a humidified atmosphere of 5% CO2. The cells were then cultured in phenol red-free medium supplemented with 5% FBS at least 24 h before actual assays.Human urothelial carcinoma cell lines, 5637, 647V, UMUC3, and TCCSUP, were originally obtained from the American Type Culture Collection  and recently authenticated by the institutional core facility. Sublines stably expressing human wild-type AR (or vector only) or AR-shRNA (or control-shRNA), including 5637-AR , 647V-AREqual amounts of proteins (30 µg) obtained from cell extracts were subjected to electrophoresis with 10% sodium dodecyl sulfate-polyacrylamide gel, which was transferred to a polyvinylidene difluoride membrane electronically. After blocking with 0.03–0.3% Blotting-Grade Blocker , the membrane was incubated with a primary antibody , GULP1 (dilution 1:100), and GAPDH (dilution 1:1000)) at 4 °C overnight, followed by 1 h incubation with a HRP-conjugated secondary antibody  at room temperature. Chemiluminescent signals were generated by a Clarity western ECL substrate and detected by ChemiDOC™ MP (Bio-Rad).https://biogrid-lasagna.engr.uconn.edu/lasagna_search/ [A ChIP assay was performed, using a Magna ChIP kit (Millipore Sigma) according to the manufacturer’s recommended protocol with minor modifications. UMUC3 cells were cross-linked with 1% formaldehyde for 10 min at room temperature. The cell lysates were sonicated in nuclear buffer . Soluble chromatin was immunoprecipitated with an anti-AR antibody and normal mouse IgG  directly conjugated with Magnetic Protein A beads. Immuno-precipitated DNA was eluted and reverse cross-linked, and DNA was extracted and purified using a spin filter column. DNA samples were analyzed by PCR. We performed a bioinformatic search  seeded in 96-well tissue culture plates were cultured for up to 96 h and then incubated with 0.5 mg/mL of MTT -2,5diphenyltetrazolium bromide, Sigma-Aldrich) for 3 h at 37 °C. MTT was dissolved by dimethyl sulfoxide, and the absorbance at 570 nm was measured. IC50 was calculated using the web-based tool .We used the MTT assay to assess cell viability. Cells , followed by counterstaining for DNA with 4′,6-diamidino-2-phenylindole. The apoptotic index was determined in the cells visualized by the fluorescence microscopy.For cell cycle phase quantification, the Cell Cycle Assay Cell-Clock™  was used according to the manufacturer’s recommended protocol. Live cells exhibit color changes associated with cell cycle phases. Data were analyzed using ImageJ version 1.53 .A scratch wound-healing assay was adapted to evaluate the ability of cell migration. Cells at a density of ≥90% confluence in 12-well tissue culture plates were scratched manually with a sterile 200 μL plastic pipette tip. The wounded monolayers of the cells were allowed to heal in serum-free medium for 24 h, and the width of the wound area was monitored with an inverted microscope. The normalized cell-free area in photographed pictures (24 h/0 h) was quantitated using ImageJ.5) in 100 μL serum-free medium were added to the upper chamber, whereas 600 µL medium containing 10% FBS was added to the lower chamber. After incubation for 24 h, invaded cells were fixed, stained with 0.1% crystal violet, and counted.Cell invasiveness was determined using a Matrigel-coated transwell chamber . Cells , as we described previously ,13,24. At-test was used to compare numerical data. Fisher’s exact test or chi-square test was used to evaluate the associations between categorized variables. Survival rates in patients were calculated by the Kaplan–Meier method and comparison was made by log-rank test. All statistical analyses were performed using Prism version 5  and EZR software [P values less than 0.05 were considered to be statistically significant.Student’s software , a graphWe identified GULP1 as a key downstream effector of AR in modulating CDDP sensitivity in bladder cancer. While GULP1 activators are not currently available, our data further support the notion that concurrent anti-androgen therapy has the potential of being a means of chemosensitization, especially in male patients with AR-positive bladder tumor. In addition, the expression status of GULP1, along with that of AR, may serve as a predictor of chemosensitivity in patients with bladder cancer.