Leaf rolling and discoloration are two chilling-injury symptoms that are widely used as indicators for the evaluation of cold tolerance at the seedling stage in rice. However, the difference in cold-response mechanism...Leaf rolling and discoloration are two chilling-injury symptoms that are widely used as indicators for the evaluation of cold tolerance at the seedling stage in rice. However, the difference in cold-response mechanisms underlying these two traits remains unknown. In the present study, a cold-tolerant rice cultivar, Lijiangxintuanheigu, and a cold-sensitive cultivar, Sanhuangzhan-2, were subjected to low-temperature treatments and physiolog-ical and genome-wide gene expression analyses were conducted. Leaf rolling occurred at temperatures lower than 11℃, whereas discoloration appeared at moderately low temperatures such as 13℃. Chlorophyll contents in both cultivars were significantly decreased at 13℃, but not altered at 11℃. In contrast, the relative water content and relative electrolyte leakage of both cultivars decreased significantly at 11℃, but did not change at 13℃. Expression of genes associated with calcium signaling and abscisic acid (ABA) degradation was significantly altered at 11℃ in comparison with 25℃ and 13℃. Numerous genes in the DREB, MYB, bZIP, NAC, Zinc finger, bHLH, and WRKY gene families were differentially expressed. Many aquaporin genes and the key genes in trehalose and starch synthesis were down regulated at 11℃ in comparison with 25℃ and 13℃. These results suggest that the two chilling injury symptoms are temperature-specific and are controlled by different mechanisms. Cold-induced leaf rolling is associated with calcium and ABA signaling pathways and is regulated by multiple transcriptional regulators. The suppression of aquaporin genes and reduced accumulation of soluble sugars under cold stress results in a reduction in cellular water potential and consequently leaf rolling.展开更多
Recent evidence exists that enoxaparin can reduce brain injury because of its anticoagulant activity. To investigate the potential therapeutic effect of enoxaparin on cold-induced traumatic brain injury, at 20 minutes...Recent evidence exists that enoxaparin can reduce brain injury because of its anticoagulant activity. To investigate the potential therapeutic effect of enoxaparin on cold-induced traumatic brain injury, at 20 minutes after modeling, male BALB/c mouse models of cold-induced traumatic brain injury were intraperitoneally administered 3 and 10 mg/kg enoxaparin or isotonic saline solution. Twenty-four hours later, enoxaparin at 10 mg/kg greatly reduced infarct volume, decreased cell apoptosis in the cortex and obviously increased serum level of total antioxidant status. By contrast, administration of enoxaparin at 3 mg/kg did not lead to these changes. These findings suggest that enoxaparin exhibits neuroprotective effect on cold-induced traumatic brain injury in a dose-dependent manner.展开更多
Pang Anshi (courtesy name:Pan Anchang,styled himself Qishui Taoist,1042-1099) was from Qizhou Prefecture (present Hubei Province).He was a famous scholar of the school of cold-induced diseases in the Northern Song Dyn...Pang Anshi (courtesy name:Pan Anchang,styled himself Qishui Taoist,1042-1099) was from Qizhou Prefecture (present Hubei Province).He was a famous scholar of the school of cold-induced diseases in the Northern Song Dynasty (960-1127).His works Supplement to the Herbals (Ben Cao Bu Yi),Pulse Study (Mai Fa),Zhu Dui Ji,etc.are all lost.His only remaining one is the General Treatise on Cold-induced Diseases (Shang Han Zong Bing Lun).展开更多
Objective:To explore the mechanism by which moxibustion improves cold-induced blood stasis pattern(CIBSP)and to uncover the biological basis underlying the correlation between the Conception Vessel(CV),Governor Vessel...Objective:To explore the mechanism by which moxibustion improves cold-induced blood stasis pattern(CIBSP)and to uncover the biological basis underlying the correlation between the Conception Vessel(CV),Governor Vessel(GV),Thoroughfare Vessel(TV),and the uterus by investigating the effects of mild moxibustion on the hypothalamic-pituitary-ovary axis(HPOA)in CIBSP model rats.Methods:Forty-eight female Sprague-Dawley rats were divided into a blank group,a model group,a CV group,a GV group,a TV group,and a non-meridian non-point(NMNP)group according to the random number table method,with 8 rats in each group.Except for rats in the blank group,the other rats were subjected to an ice water bath plus epinephrine to prepare CIBSP models.Each intervention group was given mild moxibustion on the first day of modeling.Qihai(CV6),Guanyuan(CV4),and Zhongji(CV3)were selected in the CV group;Huangshu(KI16),Dahe(KI12),and Henggu(KI11)were selected in the TV group;Xuanshu(GV5),Mingmen(GV4),and Yaoyangguan(GV3)were selected in the GV group;three NMNP spots were selected in the NMNP group.Mild moxibustion was administered for 20 min each time,once a day,for 14 consecutive days.The symptom scores of CIBSP and uterine electromyogram were compared among groups;morphological changes in reproductive organs were observed by hematoxylin-eosin staining;enzyme-linked immunosorbent assay was used to detect serum reproductive hormones and neurotransmitters.The mRNA and protein expression levels of estrogen receptor(ER),follicle-stimulating hormone receptor(FSHR),and luteinizing hormone receptor(LHR)were detected by real-time quantitative polymerase chain reaction,immunohistochemistry,or Western blotting assays.Results:Compared to the blank group,the model group showed a significantly increased CIBSP symptom score,an amplified in vivo uterine myoelectric value;decreased ovarian follicles,thinner endometrial epithelium,and disordered structure with the pathological examination,decreased serum levels of reproductive hormones,6-keto-prostaglandin F1α(6-keto-PGF1α),andβ-endorphin(β-EP),increased thromboxane B2(TXB2),5-hydroxytryptamine(5-HT),and substance P(SP),and decreased mRNA and protein expression of ovarian ER,FSHR,and LHR(P<0.05 or P<0.01).Compared to the model and NMNP groups,each point-intervention group showed a decreased CIBSP symptom score and uterine myoelectric value;increased ovarian follicles,thickened endometrium,increased glands,up-regulated serum reproductive hormones,6-keto-PGF1α,andβ-EP,down-regulated serum TXB2,5-HT,and SP and increased mRNA and protein expression of ovarian ER,FSHR,and LHR(P<0.05 or P<0.01).Conclusion:Moxibustion at points from the CV,GV,and TV(“three branches from one source”)can improve the reproductive neuroendocrine function of CIBSP rats,and this effect may be mediated through the regulation of the HPOA.These detections suggest that the biological basis of point-uterus correlation may be based on HPOA regulation.展开更多
Sympathetic arborizations act as the essential efferent signals in regulating the metabolism of peripheral organs including white adipose tissues （WAT）. However, whether these local neural structures would be of pla...Sympathetic arborizations act as the essential efferent signals in regulating the metabolism of peripheral organs including white adipose tissues （WAT）. However, whether these local neural structures would be of plastic nature, and how such plasticity might participate in specific metabolic events of WAT, remains largely uncharacterized. In this study, we exploit the new vol- ume fluorescence-imaging technique to observe the significant, and also reversible, plasticity of intra-adi- pose sympathetic arborizations in mouse inguinal WAT in response to cold challenge. We demonstrate that this sympathetic plasticity depends on the cold-elicited sig- nal of nerve growth factor （NGF） and TrkA receptor. Blockage of NGF or TrkA signaling suppresses intra- adipose sympathetic plasticity, and moreover, the cold- induced beiging process of WAT. Furthermore, we show that NGF expression in WAT depends on the cate- cholamine signal in cold challenge. We therefore reveal the key physiological relevance, together with the reg- ulatory mechanism, of intra-adipose sympathetic plas- ticity in the WAT metabolism.展开更多
Thermo-responsive shape memory hydrogels generally achieve shape fixation at low temperatures,and shape recovery at high temperatures.However,these hydrogels usually suffer from poor mechanical properties.Herein,we pr...Thermo-responsive shape memory hydrogels generally achieve shape fixation at low temperatures,and shape recovery at high temperatures.However,these hydrogels usually suffer from poor mechanical properties.Herein,we present a unique poly(acrylic acid)/calcium acetate shape memory hydrogel with cold-induced shape recovery performances as ultrastrong artificial muscles.Since the acetate groups could form aggregate at high temperatures and thus induce the association of the hydrogel network,the hydrogel can be fixed into a temporary shape upon heating and recover to its original shape in a cold environment.Moreover,a programmable shape recovery process is realized by adjusting the shape fixing time.In addition,the unique shape memory process enables the application demonstration as bio-inspired artificial muscles with an ultrahigh work density of45.2 kJ m^(-3),higher than that of biological muscles(~8 kJ m^(-3)).展开更多
A cold-induced plasma membrane protein which plays a protective role in avoiding freezing injury for some plants is one of hydrophobic proteins.A gene,named as DiRCI,was isolated from the cDNA library of Davidia invol...A cold-induced plasma membrane protein which plays a protective role in avoiding freezing injury for some plants is one of hydrophobic proteins.A gene,named as DiRCI,was isolated from the cDNA library of Davidia involucrata,containing an open reading frame of 174 bp flanked by a 92 bp 5′-untranslated sequence and a long 273 bp 3′-non-coding region.Alignment analysis indicated that the deduced amino acid sequence of 57 amino acids was high conserved with the MpRCI from Plantago asiatica,and the identity was 89.5%.Semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)analysis showed that DiRCI wasn′t detected in various organs of D.involucrata above 25℃.When the plant grew under 8℃,the gene expressed in leaves,leafstalk and seeds,but not the roots.The expression level gradually increased after 4 h of the cold treatment and reached a maximum during 24-48 h,and then decreased.The results indicated the gene did have a relation with cold stress in plant and was predicted to be one DiRCI gene.The present data not only enrich gene resources of D.involucrata,but also laid a foundation for the research on molecular mechanism of plant acclimation to cold stress.展开更多
Cold-induced sweetening(CIS)results in browning and acrylamide formation in fried potato products.β-Amylase(BAM)and vacuolar invertase(INV)are crucial for starch-sugar conversion in cold-stored potatoes.This research...Cold-induced sweetening(CIS)results in browning and acrylamide formation in fried potato products.β-Amylase(BAM)and vacuolar invertase(INV)are crucial for starch-sugar conversion in cold-stored potatoes.This research investigates the effects of salicylic acid(SA)treatment on the CiS of potatoes and gene regulation during this process.The findings revealed that the SA treatment significantly alleviated CiS by suppressing StBAM1 and St/NV1 expression in potato.Moreover,the expression of two transcription factors(TFs),StAP2/ERF and StNAC2,was inhibited by SA treatment.Transient overexpression of StAP2/ERF(AP2:APETALA2;ERF:ethylene responsive factor)and StNAC2 in tobacco leaves increased the expression of NtBAM1 and NtiNV,thus promoting starch-sugar conversion.Furthermore,the β-glucuronidase(GUS)activity demonstrated that StAP2/ERF and StNAC2 increased the promoter activity of StBAM1 and StINV1.This study identifies new TFs in CIS regulation and provides new ideas for alleviating CiIS inpotato.展开更多
Cold-inducible RNA-binding protein（CIRP）, a key regulatory protein, could be facilitated by mild hypothermia in the brain, heart and liver. This study observed the effects of mild hypothermia at 31 ± 0.5℃ on t...Cold-inducible RNA-binding protein（CIRP）, a key regulatory protein, could be facilitated by mild hypothermia in the brain, heart and liver. This study observed the effects of mild hypothermia at 31 ± 0.5℃ on traumatic brain injury in rats. Results demonstrated that mild hypothermia suppressed apoptosis in the cortex, hippocampus and hypothalamus, facilitated CIRP m RNA and protein expression in these regions, especially in the hypothalamus. The anti-apoptotic effect of mild hypothermia disappeared after CIRP silencing. There was no correlation between mitogen-activated extracellular signal-regulated kinase activation and CIRP silencing. CIRP silencing inhibited extracellular signal-regulated kinase-1/2 activation. These indicate that CIRP inhibits apoptosis by affecting extracellular signal-regulated kinase-1/2 activation, and exerts a neuroprotective effect during mild hypothermia for traumatic brain injury.展开更多
Cold-inducible RNA-binding protein （CIRP） is an RNA-binding protein that is expressed in normal testes and downregulated after heat stress caused by cryptorchidism, varicocele or environmental temperatures. The purp...Cold-inducible RNA-binding protein （CIRP） is an RNA-binding protein that is expressed in normal testes and downregulated after heat stress caused by cryptorchidism, varicocele or environmental temperatures. The purpose of this study was to investigate the functions of CIRP in the testes. We employed RNAi technique to knock down the expression of CIRP in the testes, and performed haematoxylin and eosin staining to evaluate morphological changes following knockdown. Germ cell apoptosis was examined by terminal deoxynucleotidal transferase-mediated dUTP nick end labelling （TUNEL） assay, and mitogen-activated protein kinase （MAPK） signalling pathways were investigated by Western blotting to determine the possible mechanism of apoptosis. We found that using siRNA is a feasible and reliable method for knocking down gene expression in the testes. Compared to controls, the mean seminiferous tubule diameter （MSTD） and the thickness of the germ cell layers decreased following siRNA treatment, whereas the percentage of apoptotic seminiferous tubules increased. The p44/p42, p38 and SAPK/JNK MAPK pathways were activated after downregulation of CIRP. In conclusion, we discovered that downregulation of CIRP resulted in increased germ cell apoptosis, possibly viathe activation of the p44/p42, p38 and SAPK/JNK MAPK pathways.展开更多
BACKGROUND： High incidence of stroke at interchange period of autumn and winter was demonstrated by epidemiological survey, and the specific causes should be further investigated. OBJECTIVE： To investigate the influ...BACKGROUND： High incidence of stroke at interchange period of autumn and winter was demonstrated by epidemiological survey, and the specific causes should be further investigated. OBJECTIVE： To investigate the influence of artificial cold exposure on the incidence of stroke in renovascular hypertensive rats （RHR）, and analyze the association with blood pressure and cold-inducible RNA binding protein （CIRP） mRNA expression in brain tissue. DESIGN： A completely randomized grouping design, a randomized control animal trial. SETTINGS： Lab of Neurology, the First Affiliated Hospital of Sun Yat-sen University; Department of Chemistry, Open laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, University of Hong Kong. MATERIALS： Male SD rats （n=460）, weighing 80 - 100 g were obtained from Guangdong Province Health Animal Unit. A modified RXZ-300A intelligent artificial climate cabinet （Ningbo Jiangnan Instrument Co. ,Ltd., China）. METHODS： The experiment were processed in the Lab of Neurology, the First Affiliated Hospital of Sun Yat-sen University and the Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, University of Hong Kong from October 2004 to November 2005. Rats （n = 400） were operated to establish 2-kidney 2-clip RHR model as described previously. The sham-operated rats （n =60） served as normotensive controls. Eight weeks later, 300 of RHR were randomly selected according to their systolic blood pressure （SBP） and divided into 3 sub-groups （n =100 per group）： mild hypertensive group （SBP of 160 - 200 mm Hg）, moderate hypertensive group （SBP of 200 - 220 mm Hg） and severe hypertensive group （SBP 〉 220 mm Hg）. Each group was further divided into two groups （n =50） under ACE and non-ACE. Normal sham-operated SD rats （n =60）, SBP 〈 140 mm Hg, were randomly divided into two groups： Sham-operated control group （n =30） under ACE and non-ACE. To establish the ACE and non-ACE treatment, rats were housed individually in artificial climate cabinet, and ACE was designed as three cycles of 12-hour light of 22℃ （7 ： 00 - 19 ： 00） and 12-hour dark of 4℃（19 ： 00 - 7 ： 00）. The non-ACE group was kept at 22℃ throughout the experiment. MAIN OUTCOME MEASURES： Blood Pressure changes were measured and stroke symptom were observed; Expression of the CIRP were examined by reverse transcription-polymerase chain reaction. RESULTS： Finally 360 rats were involved in the analysis of results. ①Incidence of stroke： The incidence of stroke in 2k2c RHR was significantly higher after a three-day intermittent （12-hour） ACE （29.3%） as compared with that in non-ACE （17.3%） （P 〈 0.05）. Furthermore, the severe hypertensive 2k2c RHR （BP 〉 220 mm Hg） was found to have much higher incidence of stroke （66%, 33/50） than the mild （8%, 4/50） and moderate （18%） hypertensive 2k2c RHR. ②CIRP mRNA in brain tissue： ACE treatment stimulated the mRNA expression of CIRP in non-stroke 2k2c RHR but not in stroke 2k2c RHR （P 〈 0.05）. CONCLUSION： High blood pressure and low expression of CIRP are associated with ACE induced stroke.展开更多
BACKGROUND Cold-inducible RNA-binding protein(CIRP)is related to a family of stressinduced RNA-binding proteins.It is primarily found in the nucleus,where it regulates transcription.Under stress,CIRP translocates to t...BACKGROUND Cold-inducible RNA-binding protein(CIRP)is related to a family of stressinduced RNA-binding proteins.It is primarily found in the nucleus,where it regulates transcription.Under stress,CIRP translocates to the cytoplasm where it modulates translation;a subset is secreted as extracellular CIRP(eCIRP)which is a damage-associated molecular pattern(DAMP)molecule that stimulates the production of inflammatory mediators.Elevated blood eCIRP levels may foster immune tolerance and facilitate tumor growth.Increased CIRP levels have been noted in various malignancies including colorectal cancer(CRC).This study’s objective was to determine plasma eCIRP levels before and after minimally invasive colorectal resection(MICR)for CRC.AIM To assess plasma eCIRP levels prior to and following minimally invasive colorectal resection in the context of cancer pathology.METHODS MICR patients from an IRB-approved data/tissue bank for whom plasma samples were available were eligible.Plasma specimens were obtained preoperatively(preop)and at least 3 time’s postop[between postoperative day(POD)1-41];late samples were grouped into 7-day blocks and were considered separate time points.eCIRP levels were assessed via enzyme-linked immunosorbent assay(pg/mL)and results presented as mean±SD,analysis with Wilcoxon paired t-test.RESULTS A total of 83 CRC patients who underwent MICR[colon 66%,rectal 34%;laparoscopic-assisted(LA),70%;handassisted laparoscopic(HAL),30%]were studied.The mean preop eCIRP level was 896.8±757.0 pg/mL.Elevations in mean plasma levels(P=<0.001)were noted on POD1(2549±2632 pg/mL,n=83),POD3(1871±1362 pg/mL,n=77),POD7-13(1788±1403 pg/mL,n=57),POD14-20(1473±738.8 pg/mL,n=30),and POD21-27(1681±1375 pg/mL,n=21).No significant differences were noted at POD 28-41.Higher values were noted in the HAL’s(vs LA)group,however,there were more rectal cancers in the former.CONCLUSION Elevated plasma eCIRP levels persist for a month post MICR for CRC(change from baseline,77%-184%);highest values seen on POD1.The initial surge may be due to the acute inflammatory response while later elevations may be related to wound healing and remodeling.The higher levels noted in the HAL’s group(with greater IL and more rectal cases)suggest the extent of surgical trauma impacts eCIRP levels.Further investigations are needed.展开更多
Background:Sepsis is often accompanied by lactic acidemia and acute lung injury(ALI).Clinical studies have established that high serum lactate levels are associated with increased mortality rates in septic patients.We...Background:Sepsis is often accompanied by lactic acidemia and acute lung injury(ALI).Clinical studies have established that high serum lactate levels are associated with increased mortality rates in septic patients.We further observed a significant correlation between the levels of cold-inducible RNA-binding protein(CIRP)in plasma and bronchoalveolar lavage fluid(BALF),as well as lactate levels,and the severity of post-sepsis ALI.The underlying mechanism,however,remains elusive.Methods:C57BL/6 wild type(WT),Casp8^(-/-),Ripk3^(-/-),and Zbp1^(-/-)mice were subjected to the cecal ligation and puncture(CLP)sepsis model.In this model,we measured intra-macrophage CIRP lactylation and the subsequent release of CIRP.We also tracked the internalization of extracellular CIRP(eCIRP)in pulmonary vascular endothelial cells(PVECs)and its interaction with Z-DNA binding protein 1(ZBP1).Furthermore,we monitored changes in ZBP1 levels in PVECs and the consequent activation of cell death pathways.Results:In the current study,we demonstrate that lactate,accumulating during sepsis,promotes the lactylation of CIRP in macrophages,leading to the release of CIRP.Once eCIRP is internalized by PVEC through a Toll-like receptor 4(TLR4)-mediated endocytosis pathway,it competitively binds to ZBP1 and effectively blocks the interaction between ZBP1 and tripartite motif containing 32(TRIM32),an E3 ubiquitin ligase targeting ZBP1 for proteasomal degradation.This interference mechanism stabilizes ZBP1,thereby enhancing ZBP1-receptor-interacting protein kinase 3(RIPK3)-dependent PVEC PANoptosis,a form of cell death involving the simultaneous activation of multiple cell death pathways,thereby exacerbating ALI.Conclusions:These findings unveil a novel pathway by which lactic acidemia promotes macrophage-derived eCIRP release,which,in turn,mediates ZBP1-dependent PVEC PANoptosis in sepsis-induced ALI.This finding offers new insights into the molecular mechanisms driving sepsis-related pulmonary complications and provides potential new therapeutic strategies.展开更多
Cold-inducible RNA-binding protein(CIRP) is a kind of RNA binding proteins that plays important roles in many physiological processes. The CIRP has been widely studied in mammals and amphibians since it was first clon...Cold-inducible RNA-binding protein(CIRP) is a kind of RNA binding proteins that plays important roles in many physiological processes. The CIRP has been widely studied in mammals and amphibians since it was first cloned from mammals. On the contrary, there are little reports in teleosts. In this study, the Po CIRP gene of the Japanese flounder was cloned and sequenced. The genomic sequence consists of seven exons and six introns. The putative Po CIRP protein of flounder was 198 amino acid residues long containing the RNA recognition motif(RRM). Phylogenetic analysis showed that the flounder Po CIRP is highly conserved with other teleost CIRPs. The 5' flanking sequence was cloned by genome walking and many transcription factor binding sites were identified. There is a Cp Gs region located in promoter and exon I region and the methylation state is low. Quantitative real-time PCR analysis uncovered that Po CIRP gene was widely expressed in adult tissues with the highest expression level in the ovary. The m RNA of the Po CIRP was maternally deposited and the expression level of the gene was regulated up during the gastrula and neurula stages. In order to gain the information how the protein interacts with m RNA, we performed the modeling of the 3D structure of the flounder Po CIRP. The results showed a cleft existing the surface of the molecular. Taken together, the results indicate that the CIRP is a multifunctional molecular in teleosts and the findings about the structure provide valuable information for understanding the basis of this protein's function.展开更多
Background:Extracellular cold-inducible RNA-binding protein(eCIRP)plays a vital role in the inflammatory response during cerebral ischaemia.However,the potential role and regulatory mechanism of eCIRP in traumatic bra...Background:Extracellular cold-inducible RNA-binding protein(eCIRP)plays a vital role in the inflammatory response during cerebral ischaemia.However,the potential role and regulatory mechanism of eCIRP in traumatic brain injury(TBI)remain unclear.Here,we explored the effect of eCIRP on the development of TBI using a neural-specific CIRP knockout(KO)mouse model to determine the contribution of eCIRP to TBI-induced neuronal injury and to discover novel therapeutic targets for TBI.Methods:TBI animal models were generated in mice using the fluid percussion injury method.Microglia or neuron lines were subjected to different drug interventions.Histological and functional changes were observed by immunofluorescence and neurobehavioural testing.Apoptosis was examined by a TdT-mediated dUTP nick end labelling assay in vivo or by an annexin-V assay in vitro.Ultrastructural alterations in the cells were examined via electron microscopy.Tissue acetylation alterations were identified by non-labelled quantitative acetylation via proteomics.Protein or mRNA expression in cells and tissues was determined by western blot analysis or realtime quantitative polymerase chain reaction.The levels of inflammatory cytokines and mediators in the serum and supernatants were measured via enzyme-linked immunoassay.Results:There were closely positive correlations between eCIRP and inflammatory mediators,and between eCIRP and TBI markers in human and mouse serum.Neural-specific eCIRP KO decreased hemispheric volume loss and neuronal apoptosis and alleviated glial cell activation and neurological function damage after TBI.In contrast,eCIRP treatment resulted in endoplasmic reticulum disruption and ER stress(ERS)-related death of neurons and enhanced inflammatory mediators by glial cells.Mechanistically,we noted that eCIRP-induced neural apoptosis was associated with the activation of the protein kinase RNA-like ER kinase-activating transcription factor 4(ATF4)-C/EBP homologous protein signalling pathway,and that eCIRP-induced microglial inflammation was associated with histone H3 acetylation and theα7 nicotinic acetylcholine receptor.Conclusions:These results suggest that TBI obviously enhances the secretion of eCIRP,thereby resulting in neural damage and inflammation in TBI.eCIRP may be a biomarker of TBI that can mediate the apoptosis of neuronal cells through the ERS apoptotic pathway and regulate the inflammatory response of microglia via histone modification.展开更多
基金supported in part by the Ph.D. Start-up Fund of Natural Science Foundation of Guangdong Province, China (2015A030310419)the Guangdong Scientific and Technological Plan (2015B020231002, 2017A070702006, 2017A020208022)+3 种基金the Guangzhou Scientific and Technological Plan (201804020078)the Guangdong-Hong Kong joint project (2017A050506035)the Development Project of Guangdong Provincial Key Lab (2017B030314173)the Special Fund of Central Government Guided Local Scientific Development
文摘Leaf rolling and discoloration are two chilling-injury symptoms that are widely used as indicators for the evaluation of cold tolerance at the seedling stage in rice. However, the difference in cold-response mechanisms underlying these two traits remains unknown. In the present study, a cold-tolerant rice cultivar, Lijiangxintuanheigu, and a cold-sensitive cultivar, Sanhuangzhan-2, were subjected to low-temperature treatments and physiolog-ical and genome-wide gene expression analyses were conducted. Leaf rolling occurred at temperatures lower than 11℃, whereas discoloration appeared at moderately low temperatures such as 13℃. Chlorophyll contents in both cultivars were significantly decreased at 13℃, but not altered at 11℃. In contrast, the relative water content and relative electrolyte leakage of both cultivars decreased significantly at 11℃, but did not change at 13℃. Expression of genes associated with calcium signaling and abscisic acid (ABA) degradation was significantly altered at 11℃ in comparison with 25℃ and 13℃. Numerous genes in the DREB, MYB, bZIP, NAC, Zinc finger, bHLH, and WRKY gene families were differentially expressed. Many aquaporin genes and the key genes in trehalose and starch synthesis were down regulated at 11℃ in comparison with 25℃ and 13℃. These results suggest that the two chilling injury symptoms are temperature-specific and are controlled by different mechanisms. Cold-induced leaf rolling is associated with calcium and ABA signaling pathways and is regulated by multiple transcriptional regulators. The suppression of aquaporin genes and reduced accumulation of soluble sugars under cold stress results in a reduction in cellular water potential and consequently leaf rolling.
文摘Recent evidence exists that enoxaparin can reduce brain injury because of its anticoagulant activity. To investigate the potential therapeutic effect of enoxaparin on cold-induced traumatic brain injury, at 20 minutes after modeling, male BALB/c mouse models of cold-induced traumatic brain injury were intraperitoneally administered 3 and 10 mg/kg enoxaparin or isotonic saline solution. Twenty-four hours later, enoxaparin at 10 mg/kg greatly reduced infarct volume, decreased cell apoptosis in the cortex and obviously increased serum level of total antioxidant status. By contrast, administration of enoxaparin at 3 mg/kg did not lead to these changes. These findings suggest that enoxaparin exhibits neuroprotective effect on cold-induced traumatic brain injury in a dose-dependent manner.
文摘Pang Anshi (courtesy name:Pan Anchang,styled himself Qishui Taoist,1042-1099) was from Qizhou Prefecture (present Hubei Province).He was a famous scholar of the school of cold-induced diseases in the Northern Song Dynasty (960-1127).His works Supplement to the Herbals (Ben Cao Bu Yi),Pulse Study (Mai Fa),Zhu Dui Ji,etc.are all lost.His only remaining one is the General Treatise on Cold-induced Diseases (Shang Han Zong Bing Lun).
文摘Objective:To explore the mechanism by which moxibustion improves cold-induced blood stasis pattern(CIBSP)and to uncover the biological basis underlying the correlation between the Conception Vessel(CV),Governor Vessel(GV),Thoroughfare Vessel(TV),and the uterus by investigating the effects of mild moxibustion on the hypothalamic-pituitary-ovary axis(HPOA)in CIBSP model rats.Methods:Forty-eight female Sprague-Dawley rats were divided into a blank group,a model group,a CV group,a GV group,a TV group,and a non-meridian non-point(NMNP)group according to the random number table method,with 8 rats in each group.Except for rats in the blank group,the other rats were subjected to an ice water bath plus epinephrine to prepare CIBSP models.Each intervention group was given mild moxibustion on the first day of modeling.Qihai(CV6),Guanyuan(CV4),and Zhongji(CV3)were selected in the CV group;Huangshu(KI16),Dahe(KI12),and Henggu(KI11)were selected in the TV group;Xuanshu(GV5),Mingmen(GV4),and Yaoyangguan(GV3)were selected in the GV group;three NMNP spots were selected in the NMNP group.Mild moxibustion was administered for 20 min each time,once a day,for 14 consecutive days.The symptom scores of CIBSP and uterine electromyogram were compared among groups;morphological changes in reproductive organs were observed by hematoxylin-eosin staining;enzyme-linked immunosorbent assay was used to detect serum reproductive hormones and neurotransmitters.The mRNA and protein expression levels of estrogen receptor(ER),follicle-stimulating hormone receptor(FSHR),and luteinizing hormone receptor(LHR)were detected by real-time quantitative polymerase chain reaction,immunohistochemistry,or Western blotting assays.Results:Compared to the blank group,the model group showed a significantly increased CIBSP symptom score,an amplified in vivo uterine myoelectric value;decreased ovarian follicles,thinner endometrial epithelium,and disordered structure with the pathological examination,decreased serum levels of reproductive hormones,6-keto-prostaglandin F1α(6-keto-PGF1α),andβ-endorphin(β-EP),increased thromboxane B2(TXB2),5-hydroxytryptamine(5-HT),and substance P(SP),and decreased mRNA and protein expression of ovarian ER,FSHR,and LHR(P<0.05 or P<0.01).Compared to the model and NMNP groups,each point-intervention group showed a decreased CIBSP symptom score and uterine myoelectric value;increased ovarian follicles,thickened endometrium,increased glands,up-regulated serum reproductive hormones,6-keto-PGF1α,andβ-EP,down-regulated serum TXB2,5-HT,and SP and increased mRNA and protein expression of ovarian ER,FSHR,and LHR(P<0.05 or P<0.01).Conclusion:Moxibustion at points from the CV,GV,and TV(“three branches from one source”)can improve the reproductive neuroendocrine function of CIBSP rats,and this effect may be mediated through the regulation of the HPOA.These detections suggest that the biological basis of point-uterus correlation may be based on HPOA regulation.
基金We thank members of the Zeng laboratory for helps and discussions. This work was supported by National Natural Science Foun- dation of China （Grant Nos. 31770936 and 91742106） to Wenwen Zeng, Beijing Natural Science Foundation （5172016） to Wenwen Zeng, Thousand-Talent Young Investigator Program to Wenwen Zeng, and National Key R＆D Program of China （2017YFA0505800）. The Zeng laboratory was also supported by Center for Life Sciences, Institute for Immunology, and School of Medicine at Tsinghua University.
文摘Sympathetic arborizations act as the essential efferent signals in regulating the metabolism of peripheral organs including white adipose tissues （WAT）. However, whether these local neural structures would be of plastic nature, and how such plasticity might participate in specific metabolic events of WAT, remains largely uncharacterized. In this study, we exploit the new vol- ume fluorescence-imaging technique to observe the significant, and also reversible, plasticity of intra-adi- pose sympathetic arborizations in mouse inguinal WAT in response to cold challenge. We demonstrate that this sympathetic plasticity depends on the cold-elicited sig- nal of nerve growth factor （NGF） and TrkA receptor. Blockage of NGF or TrkA signaling suppresses intra- adipose sympathetic plasticity, and moreover, the cold- induced beiging process of WAT. Furthermore, we show that NGF expression in WAT depends on the cate- cholamine signal in cold challenge. We therefore reveal the key physiological relevance, together with the reg- ulatory mechanism, of intra-adipose sympathetic plas- ticity in the WAT metabolism.
基金supported by the National Natural Science Foundation of China(51873223 and 22075154)the Natural Science Foundation of Zhejiang Province(LY19B040001)。
文摘Thermo-responsive shape memory hydrogels generally achieve shape fixation at low temperatures,and shape recovery at high temperatures.However,these hydrogels usually suffer from poor mechanical properties.Herein,we present a unique poly(acrylic acid)/calcium acetate shape memory hydrogel with cold-induced shape recovery performances as ultrastrong artificial muscles.Since the acetate groups could form aggregate at high temperatures and thus induce the association of the hydrogel network,the hydrogel can be fixed into a temporary shape upon heating and recover to its original shape in a cold environment.Moreover,a programmable shape recovery process is realized by adjusting the shape fixing time.In addition,the unique shape memory process enables the application demonstration as bio-inspired artificial muscles with an ultrahigh work density of45.2 kJ m^(-3),higher than that of biological muscles(~8 kJ m^(-3)).
基金The Key Chinese National Natrual Science Foundation(30670209)a project from Depart ment of Educationin Sichuan Province(2003A175)a Program for Science and Technology Development in Mianyang City Sichuan Province(2005BN001-1)
文摘A cold-induced plasma membrane protein which plays a protective role in avoiding freezing injury for some plants is one of hydrophobic proteins.A gene,named as DiRCI,was isolated from the cDNA library of Davidia involucrata,containing an open reading frame of 174 bp flanked by a 92 bp 5′-untranslated sequence and a long 273 bp 3′-non-coding region.Alignment analysis indicated that the deduced amino acid sequence of 57 amino acids was high conserved with the MpRCI from Plantago asiatica,and the identity was 89.5%.Semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)analysis showed that DiRCI wasn′t detected in various organs of D.involucrata above 25℃.When the plant grew under 8℃,the gene expressed in leaves,leafstalk and seeds,but not the roots.The expression level gradually increased after 4 h of the cold treatment and reached a maximum during 24-48 h,and then decreased.The results indicated the gene did have a relation with cold stress in plant and was predicted to be one DiRCI gene.The present data not only enrich gene resources of D.involucrata,but also laid a foundation for the research on molecular mechanism of plant acclimation to cold stress.
基金supported by the National Natural Science Foundation of China(No.32072281)the Youth Innovation Program of the Chinese Academy of Agricultural Sciences(No.Y2024QC12).
文摘Cold-induced sweetening(CIS)results in browning and acrylamide formation in fried potato products.β-Amylase(BAM)and vacuolar invertase(INV)are crucial for starch-sugar conversion in cold-stored potatoes.This research investigates the effects of salicylic acid(SA)treatment on the CiS of potatoes and gene regulation during this process.The findings revealed that the SA treatment significantly alleviated CiS by suppressing StBAM1 and St/NV1 expression in potato.Moreover,the expression of two transcription factors(TFs),StAP2/ERF and StNAC2,was inhibited by SA treatment.Transient overexpression of StAP2/ERF(AP2:APETALA2;ERF:ethylene responsive factor)and StNAC2 in tobacco leaves increased the expression of NtBAM1 and NtiNV,thus promoting starch-sugar conversion.Furthermore,the β-glucuronidase(GUS)activity demonstrated that StAP2/ERF and StNAC2 increased the promoter activity of StBAM1 and StINV1.This study identifies new TFs in CIS regulation and provides new ideas for alleviating CiIS inpotato.
基金supported by the National Natural Science Foundation of China,No.81303091
文摘Cold-inducible RNA-binding protein（CIRP）, a key regulatory protein, could be facilitated by mild hypothermia in the brain, heart and liver. This study observed the effects of mild hypothermia at 31 ± 0.5℃ on traumatic brain injury in rats. Results demonstrated that mild hypothermia suppressed apoptosis in the cortex, hippocampus and hypothalamus, facilitated CIRP m RNA and protein expression in these regions, especially in the hypothalamus. The anti-apoptotic effect of mild hypothermia disappeared after CIRP silencing. There was no correlation between mitogen-activated extracellular signal-regulated kinase activation and CIRP silencing. CIRP silencing inhibited extracellular signal-regulated kinase-1/2 activation. These indicate that CIRP inhibits apoptosis by affecting extracellular signal-regulated kinase-1/2 activation, and exerts a neuroprotective effect during mild hypothermia for traumatic brain injury.
文摘Cold-inducible RNA-binding protein （CIRP） is an RNA-binding protein that is expressed in normal testes and downregulated after heat stress caused by cryptorchidism, varicocele or environmental temperatures. The purpose of this study was to investigate the functions of CIRP in the testes. We employed RNAi technique to knock down the expression of CIRP in the testes, and performed haematoxylin and eosin staining to evaluate morphological changes following knockdown. Germ cell apoptosis was examined by terminal deoxynucleotidal transferase-mediated dUTP nick end labelling （TUNEL） assay, and mitogen-activated protein kinase （MAPK） signalling pathways were investigated by Western blotting to determine the possible mechanism of apoptosis. We found that using siRNA is a feasible and reliable method for knocking down gene expression in the testes. Compared to controls, the mean seminiferous tubule diameter （MSTD） and the thickness of the germ cell layers decreased following siRNA treatment, whereas the percentage of apoptotic seminiferous tubules increased. The p44/p42, p38 and SAPK/JNK MAPK pathways were activated after downregulation of CIRP. In conclusion, we discovered that downregulation of CIRP resulted in increased germ cell apoptosis, possibly viathe activation of the p44/p42, p38 and SAPK/JNK MAPK pathways.
基金the National Natural Science Foundation of China, No. 30471917the Hong Kong Research Grant Council,No. HKU 7198/01
文摘BACKGROUND： High incidence of stroke at interchange period of autumn and winter was demonstrated by epidemiological survey, and the specific causes should be further investigated. OBJECTIVE： To investigate the influence of artificial cold exposure on the incidence of stroke in renovascular hypertensive rats （RHR）, and analyze the association with blood pressure and cold-inducible RNA binding protein （CIRP） mRNA expression in brain tissue. DESIGN： A completely randomized grouping design, a randomized control animal trial. SETTINGS： Lab of Neurology, the First Affiliated Hospital of Sun Yat-sen University; Department of Chemistry, Open laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, University of Hong Kong. MATERIALS： Male SD rats （n=460）, weighing 80 - 100 g were obtained from Guangdong Province Health Animal Unit. A modified RXZ-300A intelligent artificial climate cabinet （Ningbo Jiangnan Instrument Co. ,Ltd., China）. METHODS： The experiment were processed in the Lab of Neurology, the First Affiliated Hospital of Sun Yat-sen University and the Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, University of Hong Kong from October 2004 to November 2005. Rats （n = 400） were operated to establish 2-kidney 2-clip RHR model as described previously. The sham-operated rats （n =60） served as normotensive controls. Eight weeks later, 300 of RHR were randomly selected according to their systolic blood pressure （SBP） and divided into 3 sub-groups （n =100 per group）： mild hypertensive group （SBP of 160 - 200 mm Hg）, moderate hypertensive group （SBP of 200 - 220 mm Hg） and severe hypertensive group （SBP 〉 220 mm Hg）. Each group was further divided into two groups （n =50） under ACE and non-ACE. Normal sham-operated SD rats （n =60）, SBP 〈 140 mm Hg, were randomly divided into two groups： Sham-operated control group （n =30） under ACE and non-ACE. To establish the ACE and non-ACE treatment, rats were housed individually in artificial climate cabinet, and ACE was designed as three cycles of 12-hour light of 22℃ （7 ： 00 - 19 ： 00） and 12-hour dark of 4℃（19 ： 00 - 7 ： 00）. The non-ACE group was kept at 22℃ throughout the experiment. MAIN OUTCOME MEASURES： Blood Pressure changes were measured and stroke symptom were observed; Expression of the CIRP were examined by reverse transcription-polymerase chain reaction. RESULTS： Finally 360 rats were involved in the analysis of results. ①Incidence of stroke： The incidence of stroke in 2k2c RHR was significantly higher after a three-day intermittent （12-hour） ACE （29.3%） as compared with that in non-ACE （17.3%） （P 〈 0.05）. Furthermore, the severe hypertensive 2k2c RHR （BP 〉 220 mm Hg） was found to have much higher incidence of stroke （66%, 33/50） than the mild （8%, 4/50） and moderate （18%） hypertensive 2k2c RHR. ②CIRP mRNA in brain tissue： ACE treatment stimulated the mRNA expression of CIRP in non-stroke 2k2c RHR but not in stroke 2k2c RHR （P 〈 0.05）. CONCLUSION： High blood pressure and low expression of CIRP are associated with ACE induced stroke.
文摘BACKGROUND Cold-inducible RNA-binding protein(CIRP)is related to a family of stressinduced RNA-binding proteins.It is primarily found in the nucleus,where it regulates transcription.Under stress,CIRP translocates to the cytoplasm where it modulates translation;a subset is secreted as extracellular CIRP(eCIRP)which is a damage-associated molecular pattern(DAMP)molecule that stimulates the production of inflammatory mediators.Elevated blood eCIRP levels may foster immune tolerance and facilitate tumor growth.Increased CIRP levels have been noted in various malignancies including colorectal cancer(CRC).This study’s objective was to determine plasma eCIRP levels before and after minimally invasive colorectal resection(MICR)for CRC.AIM To assess plasma eCIRP levels prior to and following minimally invasive colorectal resection in the context of cancer pathology.METHODS MICR patients from an IRB-approved data/tissue bank for whom plasma samples were available were eligible.Plasma specimens were obtained preoperatively(preop)and at least 3 time’s postop[between postoperative day(POD)1-41];late samples were grouped into 7-day blocks and were considered separate time points.eCIRP levels were assessed via enzyme-linked immunosorbent assay(pg/mL)and results presented as mean±SD,analysis with Wilcoxon paired t-test.RESULTS A total of 83 CRC patients who underwent MICR[colon 66%,rectal 34%;laparoscopic-assisted(LA),70%;handassisted laparoscopic(HAL),30%]were studied.The mean preop eCIRP level was 896.8±757.0 pg/mL.Elevations in mean plasma levels(P=<0.001)were noted on POD1(2549±2632 pg/mL,n=83),POD3(1871±1362 pg/mL,n=77),POD7-13(1788±1403 pg/mL,n=57),POD14-20(1473±738.8 pg/mL,n=30),and POD21-27(1681±1375 pg/mL,n=21).No significant differences were noted at POD 28-41.Higher values were noted in the HAL’s(vs LA)group,however,there were more rectal cancers in the former.CONCLUSION Elevated plasma eCIRP levels persist for a month post MICR for CRC(change from baseline,77%-184%);highest values seen on POD1.The initial surge may be due to the acute inflammatory response while later elevations may be related to wound healing and remodeling.The higher levels noted in the HAL’s group(with greater IL and more rectal cases)suggest the extent of surgical trauma impacts eCIRP levels.Further investigations are needed.
基金supported by grants from the USA VA(1I01BX004838 and IK6BX006297)the Shenzhen Science and Technology Program(JCYJ20230807142311024).
文摘Background:Sepsis is often accompanied by lactic acidemia and acute lung injury(ALI).Clinical studies have established that high serum lactate levels are associated with increased mortality rates in septic patients.We further observed a significant correlation between the levels of cold-inducible RNA-binding protein(CIRP)in plasma and bronchoalveolar lavage fluid(BALF),as well as lactate levels,and the severity of post-sepsis ALI.The underlying mechanism,however,remains elusive.Methods:C57BL/6 wild type(WT),Casp8^(-/-),Ripk3^(-/-),and Zbp1^(-/-)mice were subjected to the cecal ligation and puncture(CLP)sepsis model.In this model,we measured intra-macrophage CIRP lactylation and the subsequent release of CIRP.We also tracked the internalization of extracellular CIRP(eCIRP)in pulmonary vascular endothelial cells(PVECs)and its interaction with Z-DNA binding protein 1(ZBP1).Furthermore,we monitored changes in ZBP1 levels in PVECs and the consequent activation of cell death pathways.Results:In the current study,we demonstrate that lactate,accumulating during sepsis,promotes the lactylation of CIRP in macrophages,leading to the release of CIRP.Once eCIRP is internalized by PVEC through a Toll-like receptor 4(TLR4)-mediated endocytosis pathway,it competitively binds to ZBP1 and effectively blocks the interaction between ZBP1 and tripartite motif containing 32(TRIM32),an E3 ubiquitin ligase targeting ZBP1 for proteasomal degradation.This interference mechanism stabilizes ZBP1,thereby enhancing ZBP1-receptor-interacting protein kinase 3(RIPK3)-dependent PVEC PANoptosis,a form of cell death involving the simultaneous activation of multiple cell death pathways,thereby exacerbating ALI.Conclusions:These findings unveil a novel pathway by which lactic acidemia promotes macrophage-derived eCIRP release,which,in turn,mediates ZBP1-dependent PVEC PANoptosis in sepsis-induced ALI.This finding offers new insights into the molecular mechanisms driving sepsis-related pulmonary complications and provides potential new therapeutic strategies.
基金supported by the National High Technology R&D Program of China (2012AA10A402)the National Natural Science Foundation of China (31172385)
文摘Cold-inducible RNA-binding protein(CIRP) is a kind of RNA binding proteins that plays important roles in many physiological processes. The CIRP has been widely studied in mammals and amphibians since it was first cloned from mammals. On the contrary, there are little reports in teleosts. In this study, the Po CIRP gene of the Japanese flounder was cloned and sequenced. The genomic sequence consists of seven exons and six introns. The putative Po CIRP protein of flounder was 198 amino acid residues long containing the RNA recognition motif(RRM). Phylogenetic analysis showed that the flounder Po CIRP is highly conserved with other teleost CIRPs. The 5' flanking sequence was cloned by genome walking and many transcription factor binding sites were identified. There is a Cp Gs region located in promoter and exon I region and the methylation state is low. Quantitative real-time PCR analysis uncovered that Po CIRP gene was widely expressed in adult tissues with the highest expression level in the ovary. The m RNA of the Po CIRP was maternally deposited and the expression level of the gene was regulated up during the gastrula and neurula stages. In order to gain the information how the protein interacts with m RNA, we performed the modeling of the 3D structure of the flounder Po CIRP. The results showed a cleft existing the surface of the molecular. Taken together, the results indicate that the CIRP is a multifunctional molecular in teleosts and the findings about the structure provide valuable information for understanding the basis of this protein's function.
基金supported by grants from the National Natural Science Foundation of China(82172124,82130062,82241062)the National Key Research and Development Program of China(No.2022YFA1104604)the Key Medical Innovation Program of the Chinese People’s Liberation Army(18CXZ026).
文摘Background:Extracellular cold-inducible RNA-binding protein(eCIRP)plays a vital role in the inflammatory response during cerebral ischaemia.However,the potential role and regulatory mechanism of eCIRP in traumatic brain injury(TBI)remain unclear.Here,we explored the effect of eCIRP on the development of TBI using a neural-specific CIRP knockout(KO)mouse model to determine the contribution of eCIRP to TBI-induced neuronal injury and to discover novel therapeutic targets for TBI.Methods:TBI animal models were generated in mice using the fluid percussion injury method.Microglia or neuron lines were subjected to different drug interventions.Histological and functional changes were observed by immunofluorescence and neurobehavioural testing.Apoptosis was examined by a TdT-mediated dUTP nick end labelling assay in vivo or by an annexin-V assay in vitro.Ultrastructural alterations in the cells were examined via electron microscopy.Tissue acetylation alterations were identified by non-labelled quantitative acetylation via proteomics.Protein or mRNA expression in cells and tissues was determined by western blot analysis or realtime quantitative polymerase chain reaction.The levels of inflammatory cytokines and mediators in the serum and supernatants were measured via enzyme-linked immunoassay.Results:There were closely positive correlations between eCIRP and inflammatory mediators,and between eCIRP and TBI markers in human and mouse serum.Neural-specific eCIRP KO decreased hemispheric volume loss and neuronal apoptosis and alleviated glial cell activation and neurological function damage after TBI.In contrast,eCIRP treatment resulted in endoplasmic reticulum disruption and ER stress(ERS)-related death of neurons and enhanced inflammatory mediators by glial cells.Mechanistically,we noted that eCIRP-induced neural apoptosis was associated with the activation of the protein kinase RNA-like ER kinase-activating transcription factor 4(ATF4)-C/EBP homologous protein signalling pathway,and that eCIRP-induced microglial inflammation was associated with histone H3 acetylation and theα7 nicotinic acetylcholine receptor.Conclusions:These results suggest that TBI obviously enhances the secretion of eCIRP,thereby resulting in neural damage and inflammation in TBI.eCIRP may be a biomarker of TBI that can mediate the apoptosis of neuronal cells through the ERS apoptotic pathway and regulate the inflammatory response of microglia via histone modification.
