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In ischemic stroke, neutrophils are the first-line peripheral immune cells infiltrating the brain tissue to form neutrophil extracellular traps (NETs). The present study aimed to investigate the role of neuronal cold-inducible RNA-binding protein (CIRP) in promoting NETs-induced brain endothelial barrier destruction and cerebral edema after ischemic stroke. We found that the expression of NETs and neuronal CIRP in the penumbra increased at 6 hours after transient middle cerebral artery occlusion (tMCAO) and increased significantly at 24 hours, reaching a peak at 3 days. NETs degradation or CIRP inhibition can alleviate the leakage of brain endothelial barrier and reverse the decreased expression of tight junction proteins (zonula occludens-1, claudin-5 and occludin) in tMCAO mice. Oxygenglucose deprivation/reperfusion treated primary neurons or recombinant CIRP could induce NETs formation via TLR4/p38 signaling pathway in vitro. Transcription factor specificity protein 1 (sp1) was responsible for the increased neuronal CIRP expression and the inhibition of sp1 could suppress the increased CIRP expression, reduce NETs formation, and diminish brain endothelial barrier leakage in tMCAO mice. We also found the upregulated CIRP level was associated with severe cerebral edema in patients with acute ischemic stroke. In conclusion, the increased expression of transcription factor sp1 after ischemic stroke can lead to elevated CIRP expression and release from the neurons, which subsequently interacts with neutrophils and promotes NETs formation, resulting in brain endothelial barrier destruction and cerebral edema. [ABSTRACT FROM AUTHOR]

ABSTRACT: Improving immune function is an important indicator for establishing cold adaptation in broilers. In the study, to explore the effects and molecular mechanisms of intermittent and mild cold stimulation (IMCS) on the immune function of broilers, CIRP and TRPM8, induced by cold stimulation, as well as the NF-κB and MAPK pathways which play an important role in immune response, were selected to investigate. A total of 192 one-day-old broilers (Ross 308) were selected and randomly divided into the control group (CC) and the cold stimulation group (CS). The broilers in CC were raised at normal feeding temperature from d 1 to 43, while the broilers in CS were subjected to cold stimulation from day 15 to 35, with a temperature 3 °C below that of the CC group for 5 h, at 1 d intervals. The results showed that IMCS had little effect on the broiler hearts, and the myocardial structure was not damaged. On d 22, IMCS significantly increased the mRNA levels of CIRP, TRPM8, P65, P38, COX-2, TNF-α, IFN- γ, IL-6, IL-10, and the protein levels of CIRP, P65, P38, IL-1β and iNOS in the hearts, and the levels of CIRP and all cytokines in the serum (P ≤ 0.05). The mRNA and protein levels of IκB-α were significantly reduced (P ≤ 0.05). On d 36, the mRNA levels of TRPM8, P65, ERK, and IL-10 in the hearts and the content of COX-2 in the serum in CS were increased significantly (P ≤ 0.05), while the mRNA levels of IκB-α, P38, and IL-1β were decreased significantly (P ≤ 0.05). On d 43, IMCS significantly upregulated the mRNA levels of TRPM8, IFN- γ, IL-4, IL-6, IL-10, and the protein levels of IκB-α, P38, and the levels of iNOS, TNF-α, IL6 and IL10 in the serum (P ≤ 0.05); whereas it significantly downregulated CIRP, JNK, P38, iNOS, TNF-α mRNA levels, and CIRP, P65, ERK, JNK, IL1β and iNOS protein levels (P ≤ 0.05). Therefore, IMCS can enhance broiler immune function through co-regulation of CIRP and TRPM8 on the NF-κB and MAPK pathways, which facilitate the cold adaptation in broilers.

Abstract Background Downregulated expression of cold-inducible RNA binding protein (CIRP), a stress-response protein, has been demonstrated in the hearts of patients with heart failure (HF). However, whether CIRP plays a critical role in the pathogenesis of HF remains unknown. Zr17-2 is a recently identified CIRP agonist, which can enhance the expression of CIRP in hearts. Herein, we evaluated the effects of zr17-2 on the development of HF in a rat model of myocardial infarction (MI). Methods Male SD rats were pretreated with CIRP agonist zr17-2 or vehicle saline for 6 consecutive days, followed by MI induction. 1-week post-MI, cardiac function, and structural and molecular changes were determined by echocardiography and molecular biology methods. Results Excitingly, we found that pretreatment with zr17-2 significantly attenuated MI-induced cardiac dysfunction and dilation, coupled with reduced infarction size and cardiac remodeling. In addition, increased inflammatory response in the peri-infarcted heart including macrophage infiltration and the expression of inflammatory genes were all significantly decreased by zr17-2 pretreatment, suggesting an anti-inflammatory effect of zr17-2. Moreover, zr17-2 pretreatment also upregulated the antioxidant genes (e.g. NQO-1, Nrf2, and HO-1) level in the hearts. In isolated cultured cardiomyocytes, pretreatment with zr17-2 markedly attenuated cell injury and apoptosis induced by oxidative injury, along with elevation of Nrf2-related antioxidant genes and CIRP. However, silencing CIRP abolished zr17-2’s antioxidant effects against oxidative injury, confirming that zr17-2’s role is dependent on CIRP. Conclusion Collectively, our study suggests CIRP plays a crucial role in the development of HF and a beneficial effect of CIRP agonist in preventing MI-induced HF, possibly via anti-inflammatory and anti-oxidant pathways.

Background: Downregulated expression of cold-inducible RNA binding protein (CIRP), a stress-response protein, has been demonstrated in the hearts of patients with heart failure (HF). However, whether CIRP plays a critical role in the pathogenesis of HF remains unknown. Zr17-2 is a recently identified CIRP agonist, which can enhance the expression of CIRP in hearts. Herein, we evaluated the effects of zr17-2 on the development of HF in a rat model of myocardial infarction (MI). Methods: Male SD rats were pretreated with CIRP agonist zr17-2 or vehicle saline for 6 consecutive days, followed by MI induction. 1-week post-MI, cardiac function, and structural and molecular changes were determined by echocardiography and molecular biology methods. Results: Excitingly, we found that pretreatment with zr17-2 significantly attenuated MI-induced cardiac dysfunction and dilation, coupled with reduced infarction size and cardiac remodeling. In addition, increased inflammatory response in the peri-infarcted heart including macrophage infiltration and the expression of inflammatory genes were all significantly decreased by zr17-2 pretreatment, suggesting an anti-inflammatory effect of zr17-2. Moreover, zr17-2 pretreatment also upregulated the antioxidant genes (e.g. NQO-1, Nrf2, and HO-1) level in the hearts. In isolated cultured cardiomyocytes, pretreatment with zr17-2 markedly attenuated cell injury and apoptosis induced by oxidative injury, along with elevation of Nrf2-related antioxidant genes and CIRP. However, silencing CIRP abolished zr17-2's antioxidant effects against oxidative injury, confirming that zr17-2's role is dependent on CIRP. Conclusion: Collectively, our study suggests CIRP plays a crucial role in the development of HF and a beneficial effect of CIRP agonist in preventing MI-induced HF, possibly via anti-inflammatory and anti-oxidant pathways. [ABSTRACT FROM AUTHOR]

RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by associating with regulatory sequences in the untranslated regions of mRNAs. Cold-inducible RBP (CIRP) is a stress-induced RBP that was recently shown to modulate inflammation in response to cellular stress, where it increases or decreases pro-tumorigenic (proinflammatory) cytokines in different contexts. CIRP expression is altered in several cancers, including breast cancer, but the effects of CIRP on inflammation in breast cancer is not known. Here, we investigate if CIRP alters growth and the inflammatory profile of breast tumors. Transgenic mice overexpressing CIRP in the mammary epithelium were crossed with the PyMT mouse model of breast cancer, and the effects on both early and late tumorigenesis and inflammation were assessed. The effects of CIRP knockdown were also assessed in Py2T cell grafts. Overexpression of CIRP led to decreased tumorigenesis in the PyMT mouse model. Conversely, the knockdown of CIRP in Py2T cell grafts led to increased tumor growth. Luminex cytokine assays assessed the effects on the inflammatory environment. CIRP/PyMT mammary glands/mammary tumors and serum had decreased cytokines that promote inflammation, angiogenesis, and metastasis compared to PyMT mammary glands and serum, documenting a shift towards an environment less supportive of tumorigenesis. CIRP overexpression also decreased CD4+ helper T cells and increased CD8+ cytotoxic T cells in mammary tumors. Overall, these data support a role for CIRP as a potent antitumor molecule that suppresses both local and systemic pro-tumorigenic inflammation. [ABSTRACT FROM AUTHOR]

Damage-associated molecular patterns (DAMPs) play a role in the pathogenesis of vitiligo. It has been established that the cold-inducible RNA-binding protein (CIRP), a member of the family of cold-shock proteins that respond to stress, is a DAMP molecule that promotes inflammation. The objective was to evaluate the serum and tissue CIRP expression in non-segmental vitiligo (NSV) patients. A sample of 40 participants, 20 NSV patients and 20 control groups of age- and sex-matched healthy individuals were included in this case–control study where the enzyme-linked immunosorbent assay was used in detecting the serum and tissue CIRP levels in participants. The serum and tissue CIRP levels significantly increased in NSV patients compared with the healthy controls, (165.35 ± 24.42, 226.29 ± 24.00 versus 59.81 ± 12.10, 105.86 ± 11.27 pg/ml, respectively) (P < 0.01). Serum and tissue CIRP are significantly correlated with each other (r = 0.641, P = 0.002). Except for a statistically significant positive correlation between CIRP tissue level and VASI (r = 0.539, P = 0.014), the CIRP Serum and tissue did not show any statistically significant correlations with different clinical parameters in patients. ROC curve shows that the cut-off point for serum and tissue CIRP level to differentiate between patients and controls was 86.5, 124.3 pg/ml, respectively, with 100.0% sensitivity, 100.0% specificity and 1.000 AUC for each of them. It is concluded that CIRP may have a crucial role in the pathogenesis of NSV and could be used as a marker for vitiligo and its extent with the need for further large-scale study. [ABSTRACT FROM AUTHOR]

Environmental conditions regulate the growth and reproduction of fish. The increase in sea temperature during winter may have adverse effects on Takifugu rubripes. To study the mechanism of low-temperature tolerance of T. rubripes, the expression of antifreeze protein (AFP) gene, cold-induced RNA binding protein (CIRP) gene, high mobility group protein box-1 (HMGB1) gene, and Y-box binding protein (YB-1) gene in the liver, spleen, kidney, brain, heart, intestine, muscle, gonad, and skin tissues of T. rubripes obtained from different temperatures (18℃, 13℃, 8℃, and 5℃) was analyzed by quantitative real-time PCR. The results showed that the AFP gene was widely expressed in tissues, with the highest expression in the muscle (P < 0.05). With the decrease in temperature, the expression of the AFP gene in each tissue showed a significant increasing trend, reaching the highest value in the 5℃ group. The expression of the CIRP gene was the highest in the muscle (P < 0.05). With a decrease in temperature, the trend of CIRP gene expression in various tissues was different. The CIRP gene expression levels of liver, kidney, brain, heart, intestine, and skin showed a trend of initial increase, followed by a decrease, and then an increase. The expression levels in the spleen, muscle, and gonads showed an upward trend, reaching the highest value in the 5℃ group. The expression of the HMGB1 gene was the highest in muscle (P < 0.05), followed by that in the brain, liver, heart and skin. As the temperature decreased, the expression of the HMGB1 gene in all tissues except the liver increased first and then decreased, and reached the maximum value in the 8℃ group, which was significantly higher than that of the other groups (P < 0.05). The expression of the YB-1 gene was the highest in the muscle (P < 0.05), with the lowest expression level in other tissues. As the temperature decreased, the expression level of most tissues (brain, heart, intestine, kidney, liver, muscle, and spleen) increased first, then decreased, and then increased, reaching the minimum value in the 8℃ group (P < 0.05). These results show that the expression levels of the four genes are different at different temperature, reflecting the functional specificity of these four genes. Under low-temperature stress, these genes responded positively. Their expression changed to varying degrees, suggesting that the four genes may have potentially important roles in the adaptation of T. rubripes to low temperatures. In addition, by analyzing the law of gene expression, 8℃ may be the key regulatory point for T. rubripes to deal with low-temperature stress. Too low temperature may cause its regulation disorder. The results of this study can provide a relevant basis for studying the regulation mechanism of the low-temperature response of T. rubripes.

Background: Cold stress is a common environmental stress in broiler chicks. Cold-inducible RNA-binding protein (CIRP) is a conserved cold shock protein that can regulate inflammatory response through Toll-like receptor 4 (TLR4). The mechanism that how CIRP involves in the regulation of cold stress in broilers remains unclear. Methods and results: In this study, 360 7-day-old healthy male SZ901 chicks were selected and randomly allocated to four groups, and then subjected to acute cold exposure at the ambient temperature of 12 ± 1 °C for 0 h, 4 h, 8 h, and 12 h, respectively. After cold exposure, abdominall skin temperature, gene expression of CIRP–TLR4–IRE1 signaling pathway in ileum mucosa, and small intestinal structure were measured. The results showed that cold exposure decreased abdominall skin temperature, upregulated the gene expression of endoplasmic reticulum stress (ERS) markers IRE1, inflammatory factors IL-1β, IL-6, IL-10, TNF-α, and tight junction proteins ZO-1 and Occludin in ileum of chicks compared with the control group with no (0 h) cold exposure. Compared with the control group, a long time cold exposure upregulated the gene expression of CIRP, TLR4, GRP78, NF-κB in ileum mucosa, and decreased the villus height and V/C of small intestine. Conclusions: The above results suggest that acute cold stress induces endoplasmic reticulum stress via upregulating the gene expression of CIRP–TLR4–IRE1 signaling pathway, and results in the structural damage of chick intestine. [ABSTRACT FROM AUTHOR]

RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by associating with regulatory sequences in the untranslated regions of mRNAs. Cold-inducible RBP (CIRP) is a stress-induced RBP that was recently shown to modulate inflammation in response to cellular stress, where it increases or decreases pro-tumorigenic (proinflammatory) cytokines in different contexts. CIRP expression is altered in several cancers, including breast cancer, but the effects of CIRP on inflammation in breast cancer is not known. Here, we investigate if CIRP alters growth and the inflammatory profile of breast tumors. Transgenic mice overexpressing CIRP in the mammary epithelium were crossed with the PyMT mouse model of breast cancer, and the effects on both early and late tumorigenesis and inflammation were assessed. The effects of CIRP knockdown were also assessed in Py2T cell grafts. Overexpression of CIRP led to decreased tumorigenesis in the PyMT mouse model. Conversely, the knockdown of CIRP in Py2T cell grafts led to increased tumor growth. Luminex cytokine assays assessed the effects on the inflammatory environment. CIRP/PyMT mammary glands/mammary tumors and serum had decreased cytokines that promote inflammation, angiogenesis, and metastasis compared to PyMT mammary glands and serum, documenting a shift towards an environment less supportive of tumorigenesis. CIRP overexpression also decreased CD4+ helper T cells and increased CD8+ cytotoxic T cells in mammary tumors. Overall, these data support a role for CIRP as a potent antitumor molecule that suppresses both local and systemic pro-tumorigenic inflammation.

Cold-inducible RNA-binding-protein (CIRP) is a cold shock protein that plays a protective role in genotoxic stress response. CIRP modulates inflammation in human diseases, inhibits cell proliferation, and protects cells from genotoxic damage during cellular stress. The mild cold responsive element and specificity protein 1 (SP1) play a role in Cirp expression at low temperatures. Although previous studies have provided insights into the immune functions of SP1 or CIRP, the mechanisms by which CIRP and SP1 me diate inflammatory responses remain largely unknown. Therefore, in the current study, we examined whether Cirp expression is affected by genetic factors related to temperature sensitivity as well as under low temperature. We performed a genome-wide association study on cold sensitivity in 2,000 participants. Fifty-six genome-wide significant trait-locus pairs were identified (p<1×10-5, false discovery rate < 0.05). Among these variants, rs1117050 and rs11170510 had a strong linkage disequilibrium (r² > 0.8) relationship and expression quantitative trait locus-associated signals with the nearest Sp1 gene. We confirmed that the minor alleles of rs11170510 and rs58123204 were associated with increased Sp1 expression. Additionally, Sp1 overexpression led to CIRP translocation from the nucleus to the cytoplasm. CIRP protein levels increased in serum samples that had minor alleles of rs11170510 and rs58123204. Levels of various pro-inflammatory cytokines were also significantly increased in human peripheral blood mononuclear cells with minor alleles of rs11170510 and rs58123204. These results suggest that genetic factors related to cold sensitivity regulate CIRP expression and function and provide valuable insights into prediction of potential diseases through analysis of inherent genetic factors in humans. [ABSTRACT FROM AUTHOR]

Abstract Background Cold-inducible RNA binding protein (CIRP) is a newly discovered proto-oncogene. In this study, we investigated the role of CIRP in the progression of non-small cell lung cancer (NSCLC) using patient tissue samples, cultured cell lines and animal lung cancer models. Methods Tissue arrays, IHC and HE staining, immunoblotting, and qRT-PCR were used to detect the indicated gene expression; plasmid and siRNA transfections as well as viral infection were used to manipulate gene expression; cell proliferation assay, cell cycle analysis, cell migration and invasion analysis, soft agar colony formation assay, tail intravenous injection and subcutaneous inoculation of animal models were performed to study the role of CIRP in NSCLC cells; Gene expression microarray was used to select the underlying pathways; and RNA immunoprecipitation assay, biotin pull-down assay, immunopurification assay, mRNA decay analyses and luciferase reporter assay were performed to elucidate the mechanisms. The log-rank (Mantel-Cox) test, independent sample T-test, nonparametric Mann-Whitney test, Spearman rank test and two-tailed independent sample T-test were used accordingly in our study. Results Our data showed that CIRP was highly expressed in NSCLC tissue, and its level was negatively correlated with the prognosis of NSCLC patients. By manipulating CIRP expression in A549, H460, H1299, and H1650 cell lines, we demonstrated that CIRP overexpression promoted the transition of G1/G0 phase to S phase and the formation of an enhanced malignant phenotype of NSCLC, reflected by increased proliferation, enhanced invasion/metastasis and greater tumorigenic capabilities both in vitro and in vivo. Transcriptome sequencing further demonstrated that CIRP acted on the cell cycle, DNA replication and Wnt signaling pathway to exert its pro-oncogenic action. Mechanistically, CIRP directly bound to the 3′- and 5′-UTRs of CTNNB1 mRNA, leading to enhanced stability and translation of CTNNB1 mRNA and promoting IRES-mediated protein synthesis, respectively. Eventually, the increased CTNNB1 protein levels mediated excessive activation of the Wnt/β-catenin signaling pathway and its downstream targets C-myc, COX-2, CCND1, MMP7, VEGFA and CD44. Conclusion Our results support CIRP as a candidate oncogene in NSCLC and a potential target for NSCLC therapy.

Purpose: To detect miR-383-5p and cold-inducible RNA binding protein (CIRBP, CIRP) expression in patients with polycystic ovary syndrome (PCOS) and explore the mechanism underlying their effect on apoptosis in ovarian granulosa cells (GCs). Methods: GCs were extracted from follicular fluid from 101 patients. MiR-383-5p and CIRP expression were assessed by quantitative real time polymerase chain reaction analysis. Correlation between them was assessed by Spearman correlation analysis. The potential of using miR-383-5p expression for discriminating PCOS and non-PCOS patients was predicted by receiver operating characteristic curve analysis. Proliferation and apoptosis of KGN cells transfected for miR-383-5p overexpression or knockdown was evaluated using cell counting kit-8 assay, flow cytometry, and western blot analysis. CIRP was identified as a direct target of miR-383-5p, and verified by dual-luciferase reporter assay. Results: The expression level of miR-383-5p was decreased and CIRP mRNA was increased in PCOS patients. The expression of miR-383-5p was correlated negatively with body-mass index, basal luteinizing hormone and testosterone levels, luteinizing hormone/follicle-stimulating hormone ratio, and the number of retrieved and metaphase II oocytes. MiR-383-5p had sufficient potential for prediction of PCOS. There was a negative correlation between the expression of miR-383-5p and CIRP. Overexpression of miR-383-5p enhanced the apoptosis of KGN cells. CIRP reversed the effect of miR-383-5p on promotion of apoptosis. MiR-383-5p mimics could suppress the PI3K/AKT signaling pathway, which was activated by the CIRP overexpressing plasmid. Conclusions: MiR-383-5p promoted apoptosis of ovarian GCs through the PI3K/AKT signaling pathway by targeting CIRP. [ABSTRACT FROM AUTHOR]

Uncontrolled release of damage-associated molecular patterns (DAMPs) is suggested to be a major trigger for the dysregulated host immune response that leads to severe COVID-19. Cold-inducible RNA-binding protein (CIRP), is a newly identified DAMP that aggravates inflammation and tissue injury, and induces respiratory failure in sepsis. Whether CIRP contributes to the pathogenesis of respiratory failure in COVID-19 has not yet been explored. Aim: To investigate if the concentration of extracellular CIRP (eCIRP) in serum associates with respiratory failure and lung involvement by chest computed tomography (CT) in COVID-19. Methods: Herein we report a prospective observational study of patients with COVID-19 included at two University Hospitals in Sweden between April 2020 and May 2021. Serum from hospitalized patients in Örebro (N=97) were used to assess the association between eCIRP and the level of respiratory support and its correlation with pulmonary involvement on chest CT and inflammatory biomarkers. A cohort of hospitalized and non-hospitalized patients from Umeå (N=78) was used as an external validation cohort. The severity of disease was defined according to the highest degree of respiratory support; mild disease (no oxygen), non-severe hypoxemia (conventional oxygen or high-flow nasal oxygen, HFNO <50% FiO2), and severe hypoxemia (HFNO ≥50% FiO2, mechanical ventilation). Unadjusted and adjusted linear regression was used to evaluate peak eCIRP day 0-4 in respect to severity, age, sex, Charlson comorbidity score, symptom duration, and BMI. Results: Peak eCIRP concentrations were higher in patients with severe hypoxemia and were independently associated with the degree of respiratory support in both cohorts (Örebro; p=0.01, Umeå; p<0.01). The degree of pulmonary involvement measured by CT correlated with eCIRP, rs=0.30, p<0.01 (n=97). Conclusion: High serum levels of eCIRP are associated with acute respiratory failure in COVID-19. Experimental studies are needed to determine if treatments targeting eCIRP reduces the risk of acute respiratory failure in COVID-19. [ABSTRACT FROM AUTHOR]

Psoriasis is an immune-mediated skin disease with a potential morbidity in patients. Cold-inducible RNA binding protein (CIRP) is a stress responsive protein having diverse roles in cancer and inflammation. This study aimed to evaluate the expression of CIRP, (serum and tissue), in psoriasis patients and to correlate this expression to the clinico-pathological data of the patients. The serum level and tissue expression of CIRP were compared between 20 patients and 20 healthy controls. Additionally, the association between CIRP level and various clinicopathological parameters was done. The serum level of CIRP was measured by enzyme-linked immunosorbent assay (ELISA) while its tissue expression was detected via immunohistochemistry. CIRP was expressed in the epidermis of all studied cases and controls with nuclear localization. A significant difference in its epidermal expression between lesional, perilesional cases and controls was observed. It was higher in control epidermis than perilesional skin and the lowest in lesional skin. Conversely, the serum CIRP level was significantly higher in psoriasis patients compared to healthy subjects. CIRP seemed to have a significant pathologic role in psoriasis patients with evident difference in its intracellular and extracellular expression levels suggesting a potential difference it its function. [ABSTRACT FROM AUTHOR]

The innate immune system is the first line of defense against invading pathogens or sterile injuries. Pattern recognition receptors (PRR) sense molecules released from inflamed or damaged cells, or foreign molecules resulting from invading pathogens. PRRs can in turn induce inflammatory responses, comprising the generation of cytokines or chemokines, which further induce immune cell recruitment. Neutrophils represent an essential factor in the early immune response and fulfill numerous tasks to fight infection or heal injuries. The release of neutrophil extracellular traps (NETs) is part of it and was originally attributed to the capture and elimination of pathogens. In the last decade studies revealed a detrimental role of NETs during several diseases, often correlated with an exaggerated immune response. Overwhelming inflammation in single organs can induce remote organ damage, thereby further perpetuating release of inflammatory molecules. Here, we review recent findings regarding damage-associated molecular patterns (DAMPs) which are able to induce NET formation, as well as NET components known to act as DAMPs, generating a putative fatal circle of inflammation contributing to organ damage and sequentially occurring remote organ injury. [ABSTRACT FROM AUTHOR]

Uncontrolled release of damage-associated molecular patterns (DAMPs) is suggested to be a major trigger for the dysregulated host immune response that leads to severe COVID-19. Cold-inducible RNA-binding protein (CIRP), is a newly identified DAMP that aggravates inflammation and tissue injury, and induces respiratory failure in sepsis. Whether CIRP contributes to the pathogenesis of respiratory failure in COVID-19 has not yet been explored.AimTo investigate if the concentration of extracellular CIRP (eCIRP) in serum associates with respiratory failure and lung involvement by chest computed tomography (CT) in COVID-19.MethodsHerein we report a prospective observational study of patients with COVID-19 included at two University Hospitals in Sweden between April 2020 and May 2021. Serum from hospitalized patients in Örebro (N=97) were used to assess the association between eCIRP and the level of respiratory support and its correlation with pulmonary involvement on chest CT and inflammatory biomarkers. A cohort of hospitalized and non-hospitalized patients from Umeå (N=78) was used as an external validation cohort. The severity of disease was defined according to the highest degree of respiratory support; mild disease (no oxygen), non-severe hypoxemia (conventional oxygen or high-flow nasal oxygen, HFNO

Cold-inducible RNA binding protein (CIRP) is constitutively expressed at low levels across various tissues, under laying a general role for CIRP in organic adaptations to pathophysiological conditions. However, the role of CIRP in the heart remains unclear. Study the importance of the Cold-inducible RNA -binding protein as a diagnostic test of acute myocardial infraction by comparing it with high-sensitivity cardiac troponin test. This study was carried out in AL-Zahra Teaching Hospital in Iraq during the period from October 2020 to March 2021. This study was taken analytical cross-section design, included 58 patients with acute myocardial infarction, divided into two groups, the first group consist of (36) patients with AMI and positive troponin, while the second group consist of (22) patients with AMI and negative troponin, patients with acute myocardial with age range (35-90 years), in addition to 28 subjects where healthy subjects consider as control group. All patients samples were tested for CIRP test by Enzyme linked immunosorbent assay (ELISA) in order to detect antigen present in samples. Then the same study group (both case and control subjects) was used in estimation of High-sensitivity cardiac troponin by using ELISA. Patients with acute myocardial infarction showed significant decrease in serum cold-inducible RNAbinding protein (1128.08±199.15pg/ml) when compared with healthy subjects (1314.00±93.50pg/ml) with p £ 0.05. Acute myocardial patients with negative troponin level showed the least of serum level of cold-inducible RNA-binding protein (1069.62±199.35pg/ml), when compared with acute myocardial patients with positive troponin and healthy subjects. In addition, serum CIRP showed no significant correlation with the serum high sensitive troponin with p>0.05. In conclusion, through the results, it was found that there is a decrease in the level of s. serum CIRP in patient with AMI but cannot say that it is used us a substitute for diagnosis of AMI instead of troponin and it was showed little benefits when diagnosing AMI patients with negative troponin. [ABSTRACT FROM AUTHOR]

Abstract Background Neonatal sepsis and the associated myocardial dysfunction remain a leading cause of infant mortality. Extracellular cold-inducible RNA-binding protein (eCIRP) acts as a ligand of triggering receptor expressed on myeloid cells-1 (TREM-1). M3 is a small CIRP-derived peptide that inhibits the eCIRP/TREM-1 interaction. We hypothesize that the eCIRP/TREM-1 interaction in cardiomyocytes contributes to sepsis-induced cardiac dysfunction in neonatal sepsis, while M3 is cardioprotective. Methods Serum was collected from neonates in the Neonatal Intensive Care Unit (NICU). 5–7-day old C57BL/6 mouse pups were used in this study. Primary murine neonatal cardiomyocytes were stimulated with recombinant murine (rm) CIRP with M3. TREM-1 mRNA and supernatant cytokine levels were assayed. Mitochondrial oxidative stress, ROS, and membrane potential were assayed. Neonatal mice were injected with rmCIRP and speckle-tracking echocardiography was conducted to measure cardiac strain. Sepsis was induced by i.p. cecal slurry. Mouse pups were treated with M3 or vehicle. After 16 h, echocardiography was performed followed by euthanasia for tissue analysis. A 7-day survival study was conducted. Results Serum eCIRP levels were elevated in septic human neonates. rmCIRP stimulation of cardiomyocytes increased TREM-1 gene expression. Stimulation of cardiomyocytes with rmCIRP upregulated TNF-α and IL-6 in the supernatants, while this upregulation was inhibited by M3. Stimulation of cardiomyocytes with rmCIRP resulted in a reduction in mitochondrial membrane potential (MMP) while M3 treatment returned MMP to near baseline. rmCIRP caused mitochondrial calcium overload; this was inhibited by M3. rmCIRP injection impaired longitudinal and radial cardiac strain. Sepsis resulted in cardiac dysfunction with a reduction in cardiac output and left ventricular end diastolic diameter. Both were improved by M3 treatment. Treatment with M3 attenuated serum, cardiac, and pulmonary levels of pro-inflammatory cytokines compared to vehicle-treated septic neonates. M3 dramatically increased sepsis survival. Conclusions Inhibition of eCIRP/TREM-1 interaction with M3 is cardioprotective, decreases inflammation, and improves survival in neonatal sepsis. Trial registration Retrospectively registered.

Cold‐inducible RNA‐binding protein (CIRP) is a stress‐response protein that is expressed in various types of cells and acts as an RNA chaperone, modifying the stability of its targeted mRNA. Intracellular CIRP could also be released into extracellular space and once released, extracellular CIRP (eCIRP) acts as a damage‐associated molecular pattern (DAMP) to induce and amplify inflammation. Recent studies have found that eCIRP could promote acute lung injury (ALI) via activation of macrophages, neutrophils, pneumocytes and lung vascular endothelial cells in context of sepsis, haemorrhagic shock, intestinal ischemia/reperfusion injury and severe acute pancreatitis. In addition, CIRP is also highly expressed in the bronchial epithelial cells and its expression is upregulated in the bronchial epithelial cells of patients with chronic obstructive pulmonary diseases (COPD) and rat models with chronic bronchitis. CIRP is a key contributing factor in the cold‐induced exacerbation of COPD by promoting the expression of inflammatory genes and hypersecretion of airway mucus in the bronchial epithelial cells. Besides, CIRP is also involved in regulating pulmonary fibrosis, as eCIRP could directly activate and induce an inflammatory phenotype in pulmonary fibroblast. This review summarizes the findings of CIRP investigation in respiratory diseases and the underlying molecular mechanisms. [ABSTRACT FROM AUTHOR]

Extracellular cold‐inducible RNA‐binding protein (CIRP) is a proinflammatory mediator that aggravates ischaemia‐reperfusion injury (IRI). Normothermic machine perfusion (NMP) could effectively alleviate the IRI of the liver, but the underlying mechanism remains to be explored. We show that human DCD livers secreted a large amount of CIRP during static cold storage (CS), which is released into the circulation after reperfusion. The expression of CIRP was related to postoperative IL‐6 levels and liver function. In a rat model, the CIRP expression was upregulated during warm ischaemia and cold storage. Then, rat DCD livers were preserved using CS, hypothermic oxygenated machine perfusion (HOPE) and NMP. C23, a CIRP inhibitor, was administrated in the HOPE group. Compared with CS, NMP significantly inhibited CIRP expression and decreased oxidative stress by downregulating NADPH oxidase and upregulating UCP2. NMP markedly inhibited the mitochondrial fission‐related proteins Drp‐1 and Fis‐1. Further, NMP increased the mitochondrial biogenesis‐related protein, TFAM. NMP significantly reduced inflammatory reactions and apoptosis after reperfusion, and NMP‐preserved liver tissue had higher bile secretion and ICG metabolism compared to the CS group. Moreover, C23 administration attenuated IRI in the HOPE group. Additionally, HL‐7702 cells were stimulated with rhCIRP and C23. High rhCIRP levels increased oxidative stress and apoptosis. In summary, NMP attenuates the IRI of DCD liver by inhibiting CIRP‐mediated oxidative stress and mitochondrial fission. [ABSTRACT FROM AUTHOR]

Abstract Background Alcohol abuse affects the brain regions responsible for memory, coordination and emotional processing. Binge alcohol drinking has shown reductions in brain activity, but the molecular targets have not been completely elucidated. We hypothesized that brain cells respond to excessive alcohol by releasing a novel inflammatory mediator, called cold inducible RNA-binding protein (CIRP), which is critical for the decreased brain metabolic activity and impaired cognition. Methods Male wild type (WT) mice and mice deficient in CIRP (CIRP−/−) were studied before and after exposure to binge alcohol level by assessment of relative brain glucose metabolism with fluorodeoxyglucose (18FDG) and positron emission tomography (PET). Mice were also examined for object-place memory (OPM) and open field (OF) tasks. Results Statistical Parametric Analysis (SPM) of 18FDG-PET uptake revealed marked decreases in relative glucose metabolism in distinct brain regions of WT mice after binge alcohol. Regional analysis (post hoc) revealed that while activity in the temporal (secondary visual) and limbic (entorhinal/perirhinal) cortices was decreased in WT mice, relative glucose metabolic activity was less suppressed in the CIRP−/− mice. Group and condition interaction analysis revealed differing responses in relative glucose metabolism (decrease in WT mice but increase in CIRP−/− mice) after alcohol in brain regions including the hippocampus and the cortical amygdala where the percent changes in metabolic activity correlated with changes in object discrimination performance. Behaviorally, alcohol-treated WT mice were impaired in exploring a repositioned object in the OPM task, and were more anxious in the OF task, whereas CIRP−/− mice were not impaired in these tasks. Conclusion CIRP released from brain cells could be responsible for regional brain metabolic hypoactivity leading to cognitive impairment under binge alcohol conditions.

In this study, we evaluated the effects of holding in vitro-produced bovine blastocysts under mild hypothermia (33°C or 35°C), by examining viability and hatching rates of day 7 blastocysts (day 0: in vitro fertilization) cultured for 6 days and transcriptional expression of cold-inducible transcription factors Cirp and Rbm3, implicated in mild hypothermia-induced cellular protection against various types of stress. In the normothermic control (38.5°C), viability of the embryos decreased rapidly after day 10, and most samples were degenerated on day 13. However, mild hypothermia, particularly at 33°C, resulted in maintenance of high embryonic survival rates until day 13 (77.1% on day 13) and significant increases in transcriptional expression of Rbm3 in day 11 embryos compared with those at 38.5°C. Thus, our results suggested that upregulation of Rbm3 may occur in response to mild hypothermia in many bovine embryos, providing insights into the effects of mild hypothermia on embryo quality.

The main danger of cold stress to animals in cold regions is systemic metabolic changes and protein synthesis inhibition. Cold-induced RNA-binding protein is a cold shock protein that is rapidly up-regulated under cold stimulation in contrast to the inhibition of most proteins and participates in multiple cellular physiological activities by regulating targets. Therefore, this study was carried out to investigate the possible mechanism of CIRP-mediated glucose metabolism regulation and survival promotion in skeletal muscle after acute cold exposure. Skeletal muscle and serum from mice were obtained after 0, 2, 4 and 8 h of acute hypothermia exposure. Subsequently, the changes of CIRP, metabolism and apoptosis were examined. Acute cold exposure increased energy consumption, enhanced glycolysis, increased apoptosis, and up-regulated CIRP and phosphorylation of AKT. In addition, CIRP overexpression in C2C12 mouse myoblasts at each time point under 37°C and 32°C mild hypothermia increased AKT phosphorylation, enhanced glucose metabolism, and reduced apoptosis. CIRP knockdown by siRNA interference significantly reduced the AKT phosphorylation of C2C12 cells. Wortmannin inhibited the AKT phosphorylation of skeletal muscle after acute cold exposure, thereby inhibiting glucose metabolism and aggravating apoptosis. Taken together, acute cold exposure up-regulates CIRP in mouse skeletal muscle, which regulates glucose metabolism and maintains energy balance in skeletal muscle cells through the AKT signaling pathway, thus slowing down the apoptosis of skeletal muscle cells.

Background: Cold-inducible RNA binding protein (CIRP) is a newly discovered proto-oncogene. In this study, we investigated the role of CIRP in the progression of non-small cell lung cancer (NSCLC) using patient tissue samples, cultured cell lines and animal lung cancer models. Methods: Tissue arrays, IHC and HE staining, immunoblotting, and qRT-PCR were used to detect the indicated gene expression; plasmid and siRNA transfections as well as viral infection were used to manipulate gene expression; cell proliferation assay, cell cycle analysis, cell migration and invasion analysis, soft agar colony formation assay, tail intravenous injection and subcutaneous inoculation of animal models were performed to study the role of CIRP in NSCLC cells; Gene expression microarray was used to select the underlying pathways; and RNA immunoprecipitation assay, biotin pull-down assay, immunopurification assay, mRNA decay analyses and luciferase reporter assay were performed to elucidate the mechanisms. The log-rank (Mantel-Cox) test, independent sample T-test, nonparametric Mann-Whitney test, Spearman rank test and two-tailed independent sample T-test were used accordingly in our study. Results: Our data showed that CIRP was highly expressed in NSCLC tissue, and its level was negatively correlated with the prognosis of NSCLC patients. By manipulating CIRP expression in A549, H460, H1299, and H1650 cell lines, we demonstrated that CIRP overexpression promoted the transition of G1/G0 phase to S phase and the formation of an enhanced malignant phenotype of NSCLC, reflected by increased proliferation, enhanced invasion/metastasis and greater tumorigenic capabilities both in vitro and in vivo. Transcriptome sequencing further demonstrated that CIRP acted on the cell cycle, DNA replication and Wnt signaling pathway to exert its pro-oncogenic action. Mechanistically, CIRP directly bound to the 3′- and 5′-UTRs of CTNNB1 mRNA, leading to enhanced stability and translation of CTNNB1 mRNA and promoting IRES-mediated protein synthesis, respectively. Eventually, the increased CTNNB1 protein levels mediated excessive activation of the Wnt/β-catenin signaling pathway and its downstream targets C-myc, COX-2, CCND1, MMP7, VEGFA and CD44. Conclusion: Our results support CIRP as a candidate oncogene in NSCLC and a potential target for NSCLC therapy. [ABSTRACT FROM AUTHOR]

Extracellular cold‐inducible RNA‐binding protein (eCIRP) is a damage‐associated molecular pattern. Neutrophils present in the mononuclear cell fraction of Ficoll gradient separation are called low‐density neutrophils (LDNs). Here we report the novel role of eCIRP on LDNs' heterogeneity in sepsis. Sepsis was induced in male C57BL/6 wild‐type (WT) and CIRP−/− mice by cecal ligation and puncture (CLP). At 20 h after CLP, LDNs in the blood were isolated by Ficoll gradient separation, followed by staining the cells with anti‐Ly6G and anti‐CD11b Abs and detection by flow cytometry. Sepsis or recombinant murine CIRP (rmCIRP) injection in mice resulted in significant increase in the frequency (%) and number of Ly6G+CD11bhi and Ly6G+CD11blo LDNs in the blood compared to sham‐ or vehicle‐treated mice. At 20 h of CLP, CIRP−/− mice had significantly lower frequency and number of Ly6G+CD11bhi and Ly6G+CD11blo LDNs in the blood compared to WT mice. In sepsis mice or rmCIRP‐injected mice, compared to Ly6G+CD11blo LDNs, the expression of CXCR4, ICAM‐1, and iNOS and formation of reactive oxygen species, and neutrophil extracellular traps in Ly6G+CD11bhi LDNs in the blood were significantly increased. Treatment of WT bone marrow‐derived neutrophils (BMDNs) with rmCIRP increased Ly6G+CD11bhi LDN frequency, whereas treatment of TLR4−/− BMDNs with rmCIRP significantly decreased the frequency of Ly6G+CD11bhi LDNs. BMDNs' stimulation with rmCIRP increased the expression of transcription factors in LDNs. eCIRP induces the formation of a proinflammatory phenotype Ly6G+CD11bhi of LDNs through TLR4. Targeting eCIRP may provide beneficial outcomes in sepsis by decreasing proinflammatory Ly6G+CD11bhi LDNs. [ABSTRACT FROM AUTHOR]

Objective: Severe acute pancreatitis (SAP) can lead to acute lung injury (ALI). This study investigated the therapeutic effect of emodin and its molecular mechanisms in a rat model of SAP-ALI.Methods: Forty male Sprague-Dawley rats were randomly divided into the groups: Control (CON), SAP (SAP), emodin (EMO), and C23 (C23). The latter three groups of rats were induced for SAP-ALI by retrograde injection of 5% sodium taurocholate into the biliary-pancreatic duct and were treated with vehicle, emodin or C23, respectively. One day post induction, their pancreatic and lung injury was assessed by histology and arterial blood gas analysis. In vitro, rat alveolar macrophages (NR8383 cells) were treated with recombinant rat CIRP in the presence or absence of TAK242 (a TLR4 inhibitor), C23 or emodin. The CIRP-mediated activation of the NLRP3/IL-1β/CXCL1 signaling in rat lungs and NR8383 cells was determined. Similarly, the role of IL-1β in the CIRP-induced CXCL1 expression was investigated.Results: Emodin treatment significantly reduced inflammation and tissue damages in the pancreatic and lung tissues in rats with SAP-ALI, accompanied by decreasing serum amylase, CIRP and IL-1β levels and improving lung function. Furthermore, emodin significantly mitigated the SAP-up-regulated CIRP expression in the pancreatic islets and lung tissues, and attenuated the SAP-activated NF-κB signaling, NLRP3 inflammasome formation and CXCL1 expression in lung resident macrophages as well as neutrophil infiltration in the lungs of rats. In addition, treatment with CIRP significantly activated the NF-κB signaling and NLRP3 inflammasome formation and induced IL-1β and CXCL1 expression and pyroptosis in NR8383 cells, which were abrogated by TAK242 and significantly mitigated by C23 or emodin. Moreover, CIRP only induced very lower levels of CXCL1 expression in IL-1β-silencing NR8383 cells and treatment with IL-1β induced CXCL1 expression in NR8383 cells in a dose and time-dependent manner.Conclusion: Emodin may inhibit the CIRP-activated NLRP3/IL-1β/CXCL1signaling to decrease neutrophil infiltration and ameliorate the SAP-ALI in rats.

To investigate the effects of RNA‐binding proteins cold‐inducible RNA binding protein (CIRP) and human antigen R (HuR) on expression of Claudin1 and mucosal barrier function in ulcerative colitis (UC). The clinical specimens of UC patients and healthy volunteers were collected. In the clinical experiments, the expressions of CIRP, Claudin1, and HuR, along with their correlations in tissues of UC patients were analyzed by qRT‐PCR, Western blot and Pearson correlation coefficient, respectively. The chi‐square test was utilized to assess the relevance between CIRP/HuR/Claudin1 level and clinicopathological characteristics of UC patients. The in vitro and in vivo models of UC were established by lipopolysaccharide treatment or dextran sulfate sodium injection. For cell experiments, after loss‐ and gain‐of‐function, the roles of CIRP or HuR in the apoptosis and proliferation of enterocytes were examined by flow cytometry and CCK‐8 assay. The intestinal epithelial barrier function was inspected after determination on transepithelial electrical resistance value, horseradish peroxidase permeability and expressions of tight junction proteins (Occludin, ZO‐1, and JAM‐1). The relationship between HuR, CIRP, and Claudin1 was performed by RNA immunoprecipitation and dual‐luciferase reporter gene assay. For in vivo experiments, the disease activity index score, weight loss and colon length of mice were assessed to observe the effect of CIRP or HuR on the UC mouse models. Histological analysis of colon tissues was conducted by H&E staining. FITC‐dextran tracking was applied to inspect the intestinal mucosal barrier function of UC mouse models. In this study, high expression of CIRP and low expressions of HuR and Claudin1 were observed in patients, cells and mouse models of UC. The expressions of CIRP, HuR, and Claudin1 were correlated with the severity of patients with UC. There was a negative correlation between CIRP and Claudin1, and as a positive correlation between HuR and Claudin1. Claudin1 can be suppressed by CIRP, while enhanced by HuR. HuR and CIRP can competitively bind to Claudin1. HuR upregulation or CIRP downregulation promoted proliferation, suppressed apoptosis and ameliorated the damage of the barrier function in enterocytes. The in vivo experiments verified that the ameliorated damage of the intestinal mucosal barrier function in UC mice occurred with HuR overexpression or CIRP knockdown. CIRP and HuR confer pivotal effect on the intestinal mucosal barrier function of UC through competitively binding to Claudin1 mRNA. [ABSTRACT FROM AUTHOR]

Cold-inducible RNA-binding protein (CIRP) is a versatile RNA-binding protein, pivotal in modulating cellular responses to diverse stress stimuli including cold shock, ultraviolet radiation, hypoxia, and infections, with a principal emphasis on cold stress. The temperature range of 32-34 °C is most suitable for CIRP expression. The human CIRP is an 18-21 kDa polypeptide containing 172 amino acids coded by a gene located on chromosome 19p13.3. CIRP has an RNA-recognition motif (RRM) and an arginine-rich motif (RGG), both of which have roles in coordinating numerous cellular activities. CIRP itself also undergoes conformational changes in response to diverse environmental stress. Transcription factors such as hypoxia-inducible factor 1 alpha and nuclear factor-kappa B have been implicated in coordinating CIRP transcription in response to specific stimuli. The potential of CIRP to relocate from the nucleus to the cytoplasm upon exposure to different stimuli enhances its varied functional roles across different cellular compartments. The different functions include decreasing nutritional demand, apoptosis suppression, modulation of translation, and preservation of cytoskeletal integrity at lower temperatures. This review explores the diverse functions and regulatory mechanisms of CIRP, shedding light on its involvement in various cellular processes and its implications for human health and disease., Competing Interests: Declarations of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cold-inducible RNA-binding protein (CIRP) is an intracellular stress-response protein that can respond to various stress conditions by changing its expression and regulating mRNA stability. As an RNA-binding protein, CIRP modulates gene expression at the post-transcriptional level, including those genes involved in DNA repair, cellular redox metabolism, circadian rhythms, telomere maintenance, and cell survival. CIRP is expressed in a large variety of tissues, including testis, brain, lung, kidney, liver, stomach, bone marrow, and heart. Recent studies have observed the important role of CIRP in cardiac physiology and diseases. CIRP regulates cardiac electrophysiological properties such as the repolarization of cardiomyocytes, the susceptibility of atrial fibrillation, and the function of the sinoatrial node in response to stress. CIRP has also been suggested to protect cardiomyocytes from apoptosis under various stress conditions, including heart failure, high glucose conditions, as well as during extended heart preservation under hypothermic conditions. This review summarizes the findings of CIRP investigations in cardiac physiology and diseases and the underlying molecular mechanism.

Purpose: To investigate the effect of anti-cold induced RNA binding protein (CIRP) antibody on inflammation, tumor formation and abdominal aortic aneurysm in rats. Methods: Thirty healthy male Wistar rats were assigned to pseudo-operation, abdominal aortic aneurysm model, and anti-CIRP groups, with 10 in each group. The levels of CIRP, TNF-α, monocyte giant cytokine chemokine-1 (MCP-1), Toll-like receptor 4 (TLR4)) and nuclear factor kappaB (NF-κB) were determined compared among the groups. Results: At both 2 and 4 weeks, the expression of CIRP protein in the model group was significantly higher than that in the sham operation group (p < 0.05). At these two time-points, tumor formation and maximum diameter were higher in anti-CIRP and model control rats than in pseudo-operation rats. After 4 weeks of treatment, the protein expressions of TNF-α, MCP-1, TLR4 and NF-κB were higher in anti-CIRP and model control rats than in pseudo-operation rats, but were lower than model control values (p < 0.05). Conclusion: CIRP expression is significantly increased in abdominal aortic aneurysm tissue and serum, and is involved in the onset and progress of abdominal aortic aneurysm. Anti-CIRP antibody therapy effectively suppresses tumorigenesis, and inhibits tumor wall inflammatory reaction via TLR4/NF-κB pathway. This finding provides a clue and new strategy for the clinical management of abdominal aortic aneurysm. [ABSTRACT FROM AUTHOR]

• Sustaining high serum CIRP level was strongly associated with increased Treg/Th17 ratio in late-stage septic patients. • CIRP facilitates Treg expansion during immunosuppressive the phase of murine sepsis model. • CIRP acts as a proliferation signal for activated T lymphocytes. • CIRP promotes Treg cells and inhibits development of Th17 cells through TLR4-IL-2 signaling in vitro. T helper (Th) cell imbalances have been associated with the pathophysiology of sepsis, including the Th1/Th2 and Th17/T regulatory cells (Treg) paradigms. Cold-inducible RNA-binding protein (CIRP), a novel damage-associated molecular pattern (DAMP) was reported that could induce T cell activation, and skew CD4+ T cells towards a Th1 profile. However, the effect and underlying mechanisms of CIRP on Th17/Treg differentiation in sepsis still remains unknown. A prospective exploratory study including patients with sepsis was conducted. Blood samples were collected from patients on days 0, 3 and 7 on admission. The serum CIRP and peripheral blood Treg/Th17 percentage was determined by ELISA and flow cytometry. CD4+ T cells from the spleen and lymph nodes of mice with experimental sepsis were collected after treatment with normal saline (NS), recombinant murine CIRP (rmCIRP) and C23 (an antagonist for CIRP-TLR4) at late stage of sepsis. RNA-seq was conducted to reveal the pivotal molecular mechanism of CIRP on Treg/Th17 differentiation. Naïve CD4+ T cell was isolated from the Tlr4 null and wildtype mice in the presence or absence rmCIRP and C23 to confirmed above findings. A total of 19 patients with sepsis finally completed the study. Serum CIRP levels remained high in the majority of patients up to 1 week after admittance was closely associated with high Treg/Th17 ratio of peripheral blood and poor outcome. A univariate logistic analysis demonstrated that higher CIRP concentration at Day 7 is an independent risk factor for Treg/Th17 ratio increasing. CIRP promotes Treg development and suppresses Th17 differentiation was found both in vivo and in vitro. Pretreated with C23 not only alleviated the majority of negative effect of CIRP on Th17 differentiation, but also inhibited Treg differentiation, to some extent. Tlr4 deficiency could abolish almost all downstream effects of rmCIRP. Furthermore, IL-2 is proved a key downstream molecules of the effect CIRP, which also could amplify the activated CD4+ T lymphocytes. Persistent high circulating CIRP level may lead to Treg/Th17 ratio elevated through TLR4 and subsequent active IL-2 signaling which contribute to immunosuppression during late phases of sepsis. [ABSTRACT FROM AUTHOR]

Neural stem cells (NSCs) reside physiologically in a hypoxic niche to maintain self-renewal and multipotency. Whereas mild hypoxia is known to promote NSC proliferation, severe hypoxia in pathological conditions exerts the reverse effect. The multi-functional RNA-binding protein RBM3 is abundant in NSCs and can be regulated by hypoxic exposure. Although RBM3 has been shown to accelerate cell growth in many cell types, whether and how it affects NSC proliferation in hypoxic environment remains largely unknown. In this study, we tested how RBM3 regulates cell proliferation under hypoxia in C17.2 mouse NSC cell line and in primary mouse NSCs from both the forebrain of postnatal day 0 (P0) mice and the subgranular zone (SGZ) of adult mice. Our results demonstrated that RBM3 expression was highly sensitive to hypoxia, and NSCs were arrested in G0/G1 phase by 5, 2.5, and 1% O2 treatment. When we overexpressed RBM3, hypoxia-induced cell cycle arrest in G0/G1 phase was relieved and more cell transit into S phase was observed. Furthermore, cell viability under hypoxia was also increased by RBM3. In contrast, in RBM3-depleted primary NSCs, less BrdU-incorporated cells were detected, indicating exacerbated cell cycle arrest in G1 to S phase transition. Instead, overexpressed RBM3 significantly increased proliferation ratio in primary NSCs. Our findings indicate RBM3 as a potential target to maintain the proliferation capacity of NSCs under hypoxia, which can be important in NSC-based therapies of acute brain injury and chronic neurodegenerative diseases.

Sepsis is a deadly inflammatory syndrome caused by an exaggerated immune response to infection. Much has been focused on host response to pathogens mediated through the interaction of pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs). PRRs are also activated by host nuclear, mitochondrial, and cytosolic proteins, known as damage-associated molecular patterns (DAMPs) that are released from cells during sepsis. Some well described members of the DAMP family are extracellular cold-inducible RNA-binding protein (eCIRP), high mobility group box 1 (HMGB1), histones, and adenosine triphosphate (ATP). DAMPs are released from the cell through inflammasome activation or passively following cell death. Similarly, neutrophil extracellular traps (NETs) are released from neutrophils during inflammation. NETs are webs of extracellular DNA decorated with histones, myeloperoxidase, and elastase. Although NETs contribute to pathogen clearance, excessive NET formation promotes inflammation and tissue damage in sepsis. Here, we review DAMPs and NETs and their crosstalk in sepsis with respect to their sources, activation, release, and function. A clear grasp of DAMPs, NETs and their interaction is crucial for the understanding of the pathophysiology of sepsis and for the development of novel sepsis therapeutics.

The prevalence of peripheral nerve injury has attracted increased attention. Allografting has been proposed as a potential treatment strategy for peripheral nerve injury. Moreover, cryopreservation may provide almost unlimited graft material. We investigated whether cold‐inducible RNA‐binding protein (CIRP) could protect peripheral nerves during cryopreservation to promote regeneration postoperation. First, CIRP was highly expressed after pretreatment at 32°C. After 4 weeks of cryopreservation, the increased live cells, low Bax/Bcl‐2 ratio and high nerve growth factor and glial cell‐derived neurotrophic factor levels in the 32°C group demonstrated high nerve graft viability. At 4 weeks postoperation, 32°C‐Allo group demonstrated low plasma levels of interleukin‐6 and interferon‐gamma and a diminished cellular immune response. At 20 weeks postoperation, nerve regeneration in the 32°C‐Allo group was similar to that in the fresh isograft group and superior to that in the 4°C‐Allo and 15°C‐Allo groups. Moreover, the compound muscle action potential and the motor nerve conduction velocity of the 32°C‐Allo group were equal to those of the fresh isograft group. In conclusion, CIRP induction increased Schwann cell biological activity, inhibited cell apoptosis, reduced immune rejection, and promoted recipient nerve regeneration. Thus, CIRP could exert protective effects during nerve storage and stimulate regeneration in peripheral nerve reconstruction. [ABSTRACT FROM AUTHOR]

Sepsis is a deadly inflammatory syndrome caused by an exaggerated immune response to infection. Much has been focused on host response to pathogens mediated through the interaction of pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs). PRRs are also activated by host nuclear, mitochondrial, and cytosolic proteins, known as damage-associated molecular patterns (DAMPs) that are released from cells during sepsis. Some well described members of the DAMP family are extracellular cold-inducible RNA-binding protein (eCIRP), high mobility group box 1 (HMGB1), histones, and adenosine triphosphate (ATP). DAMPs are released from the cell through inflammasome activation or passively following cell death. Similarly, neutrophil extracellular traps (NETs) are released from neutrophils during inflammation. NETs are webs of extracellular DNA decorated with histones, myeloperoxidase, and elastase. Although NETs contribute to pathogen clearance, excessive NET formation promotes inflammation and tissue damage in sepsis. Here, we review DAMPs and NETs and their crosstalk in sepsis with respect to their sources, activation, release, and function. A clear grasp of DAMPs, NETs and their interaction is crucial for the understanding of the pathophysiology of sepsis and for the development of novel sepsis therapeutics. [ABSTRACT FROM AUTHOR]

Neonatal sepsis remains a leading cause of infant mortality. Cold-inducible RNA binding protein (CIRP) is an inflammatory mediator that induces TNF-α production in macrophages. C23 is a CIRP-derived peptide that blocks CIRP from binding its receptor. We therefore hypothesized that treatment with C23 reduces systemic inflammation and protects the lungs in neonatal sepsis. Sepsis was induced in C56BL/6 mouse pups (5–7 days) by intraperitoneal injection of adult cecal slurry (0.525 mg/g body weight, LD 100). One hour later pups received retroorbital injection of C23 (8 mg/kg) or vehicle (normal saline). Ten hours after sepsis induction, blood and tissues were collected for analysis. C23 treatment resulted in a 58% and 69% reduction in serum levels of proinflammatory cytokines IL-6 and IL-1β, respectively, and a 40% and 45% reduction of AST and LDH, as compared to vehicle-treated septic pups. In the lungs, C23 treatment reduced expression of cytokines IL-6 and IL-1β by 78% and 74%. In addition, the mRNA level of neutrophil chemoattractants KC and MIP-2 was reduced by 84% and 74%, respectively. These results corresponded to a reduction in histologic lung injury score. Vehicle-treated pups scored 0.49 ± 0.19, while C23 treatment reduced scores to 0.29 ± 0.12 (p < 0.05; Max = 1). Apoptosis in the lungs, measured by TUNEL assay, was also decreased by 53% with C23 treatment (p < 0.05). Inhibition of CIRP with C23 treatment is protective in septic neonatal mice as demonstrated by reduced inflammatory markers systemically and in the lung. Therefore, C23 has promising therapeutic potential in treatment of neonatal sepsis. Level I. [ABSTRACT FROM AUTHOR]

The role of cold inducible RNA-binding protein (CIRP) in mediating ischemic brain injury in neonatal rats under chronic hypobaric hypoxia was investigated. The neonatal rat model of chronic hypobaric hypoxia and the cell culture model of SH-SY5Y cells exposed to hypoxia (1% O2) were constructed. The expression of CIRP and hypoxia-inducible factor-1α (HIF-1α) was detected after hypoxic exposure, and the apoptosis-related proteins were analyzed via terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and western blot analysis to detect neuronal apoptosis. Moreover, the effects of CIRP overexpression on HIF-1α and neuronal apoptosis were identified. Chronic hypobaric hypoxia can lead to HIF-1α expression and neuronal apoptosis in the body. CIRP was induced at early exposure (3 d/7 d). However, the CIRP level in the hypoxic group was obviously lower than that in the control group with the prolongation of exposure time (21 d). In addition, the knockdown of HIF-1α significantly reduced the neuronal apoptosis under hypoxic conditions, indicating that HIF-1α may promote apoptosis during exposure. The overexpression of CIRP significantly inhibited the upregulation of HIF-1α during hypoxia and the HIF-1α-mediated neuronal apoptosis. Results of the current study showed that, CIRP is involved in the ischemic brain injury induced by chronic hypoxia through downregulation of HIF-1α expression. [ABSTRACT FROM AUTHOR]

Cold‐inducible RNA‐binding protein (CIRP) was discovered 2 decades ago while studying the mechanism of cold stress adaptation in mammals. Since then, the role of intracellular CIRP (iCIRP) as a stress‐response protein has been extensively studied. Recently, extracellular CIRP (eCIRP) was discovered to also have an important role, acting as a damage‐associated molecular pattern, raising critical implications for the pathobiology of inflammatory diseases. During hemorrhagic shock and sepsis, inflammation triggers the translocation of CIRP from the nucleus to the cytosol and its release to the extracellular space. eCIRP then induces inflammatory responses in macrophages, neutrophils, lymphocytes, and dendritic cells. eCIRP also induces endoplasmic reticulum stress and pyroptosis in endothelial cells by activating the NF‐κB and inflammasome pathways, and necroptosis in macrophages via mitochondrial DNA damage. eCIRP works through the TLR4‐MD2 receptors. Studies with CIRP−/− mice reveal protection against inflammation, implicating eCIRP to be a novel drug target. Anti‐CIRP Ab or CIRP‐derived small peptide may have effective therapeutic potentials in sepsis, acute lung injury, and organ ischemia/reperfusion injuries. The current review focuses on the pathobiology of eCIRP by emphasizing on signal transduction machineries, leading to discovering novel therapeutic interventions targeting eCIRP in various inflammatory diseases. [ABSTRACT FROM AUTHOR]

The response of cold shock proteins to exercise and environmental temperature in human skeletal muscle is not known. The purpose of this study was to determine the early mRNA response of human stress proteins to endurance exercise and environmental temperatures. Seven recreationally trained males cycled for 1 hour at 60% VO2peak in 7°C, 20°C, and 33°C with biopsies taken pre- and 3 hours post-exercise. Gene expression for heat shock and cold shock proteins were analyzed using qRT-PCR on muscle biopsy samples from the vastus lateralis. RBM3 mRNA was reduced 1.43 ± 0.10 fold (p = 0.006) while there was a trend for CIRP to decrease1.27 ± 0.14 fold (p = 0.059) from pre- to 3 h post-exercise. CIRP and RBM3 mRNA were not different between temperatures (p = 0.273 and p = 0.686, respectively). HSP70 mRNA was 2.27 ± 0.23 fold higher 3 h post-exercise when compared to pre-exercise (p = 0.002) but was not significantly different between temperatures (p = 0.103). HSP27, HSP90, and HSF1 mRNA did not change from pre- to post-exercise (p = 0.052, p = 0.324, p = 0.795) and were not different between temperatures (p = 0.247, p = 0.134, p = 0.808). These data indicate that exposure to mild heat and cold during aerobic exercise have limited effect on the skeletal muscle mRNA expression of heat shock and cold shock proteins. However, skeletal muscle mRNA of cold shock proteins decrease, while HSP70 mRNA increases in response to a low to moderate intensity aerobic exercise bout. [ABSTRACT FROM AUTHOR]

Cold-inducible RNA-binding protein (CIRP) is an intracellular RNA-chaperone and extracellular promoter of inflammation, which is increasingly expressed and released under conditions of hypoxia and cold stress. The functional relevance of CIRP for angiogenesis and regeneration of ischemic muscle tissue has never been investigated and is the topic of the present study. We investigated the role of CIRP employing CIRP deficient mice along with a hindlimb model of ischemia-induced angiogenesis. 1 and 7 days after femoral artery ligation or sham operation, gastrocnemius muscles of CIRP-deficient and wildtype mice were isolated and processed for (immuno-) histological analyses. CIRP deficient mice showed decreased ischemic tissue damage as evidenced by Hematoxylin and Eosin staining, whereas angiogenesis was enhanced as demonstrated by increased capillary/muscle fiber ratio and number of proliferating endothelial (CD31+/BrdU+) cells on day 7 after surgery. Moreover, CIRP deficiency resulted in a reduction of total leukocyte count (CD45+), neutrophils (myeloperoxidase, MPO+), neutrophil extracellular traps (NETs) (MPO+/CitH3+), and inflammatory M1-like polarized macrophages (CD68+/MRC1-), whereas the number of tissue regenerating M2-like polarized macrophages (CD68+/MRC1-) was increased in ischemic tissue samples. In summary, we show that the absence of CIRP ameliorates angiogenesis and regeneration of ischemic muscle tissue, most likely by influencing macrophage polarization in direction to regenerative M2-like macrophages.

• CIRP induces proliferation, migration, invasion and inflammation of RA-FLSs. • Blocking of CIRP with C23 could inhibit CIRP-induced abnormal activation of RA-FLSs. • C23 could alleviate the arthritis severity in AA rats. • Knockdown of TLR4 inhibit CIRP-induced abnormal activation of RA-FLSs. • CIRP induces abnormal activation of RA-FLSs via TLR4-mediated HDAC3. The development of rheumatoid arthritis (RA) is closely related to the excessive activation of fibroblast-like synoviocytes (FLSs), which are regulated by a variety of endogenous proinflammatory molecules. Extracellular cold-inducible RNA-binding protein (CIRP), as a novel endogenous proinflammatory molecule, plays an important role in inflammatory diseases. More importantly, the synovial concentration of CIRP in patients with RA was significantly higher than that in patients with osteoarthritis (OA). Thus, this study aimed to investigate the role of extracellular CIRP in the abnormal activation of RA-FLSs and its related mechanisms. Our study showed that extracellular CIRP induced proliferation, migration and invasion of RA-FLSs, increased the expression of N-cadherin and MMP-3, and promoted the release of IL-1β and IL-33. However, blocking of extracellular CIRP with C23 inhibited CIRP-induced abnormal activation of RA-FLSs and alleviated the arthritis severity in AA rats. Accumulating evidence suggests that the activity and proinflammatory effects of CIRP are mediated through Toll-like receptor 4 (TLR4). Further studies demonstrated that the TLR4 knockdown inhibited CIRP-induced abnormal activation, and histone deacetylase 3 (HDAC3) expression in RA-FLSs. In addition, we found that HDAC3 knockdown and the specific inhibitor RGFP966 significantly suppressed CIRP-induced abnormal activation of RA-FLSs. We further found that treatment with HDAC3 specific inhibitor effectively alleviated the severity of arthritis in AA rats. Taken together, these findings indicate that extracellular CIRP induces abnormal activation of RA-FLSs via the TLR4-mediated HDAC3 pathways. [ABSTRACT FROM AUTHOR]

Abstract: Sepsis represents uncontrolled inflammation due to an infection. Cold‐inducible RNA‐binding protein (CIRP) is a stress‐induced damage‐associated molecular pattern (DAMP). A subset of neutrophils expressing ICAM‐1+ neutrophils was previously shown to produce high levels of reactive oxygen species. The role of CIRP for the development and function of ICAM‐1+ neutrophils during sepsis is unknown. We hypothesize that CIRP induces ICAM‐1 expression in neutrophils causing injury to the lungs during sepsis. Using a mouse model of cecal ligation and puncture (CLP)‐induced sepsis, we found increased expression of CIRP and higher frequencies and numbers of ICAM‐1+ neutrophils in the lungs. Conversely, the CIRP−/− mice showed significant inhibition in the frequencies and numbers of ICAM‐1+ neutrophils in the lungs compared to wild‐type (WT) mice in sepsis. In vitro treatment of bone marrow‐derived neutrophils (BMDN) with recombinant murine CIRP (rmCIRP) significantly increased ICAM‐1+ phenotype in a time‐ and dose‐dependent manner. The effect of rmCIRP on increasing frequencies of ICAM‐1+ neutrophils was significantly attenuated in BMDN treated with anti‐TLR4 Ab or NF‐κB inhibitor compared, respectively, with BMDN treated with isotype IgG or DMSO. The frequencies of iNOS producing and neutrophil extracellular traps (NETs) forming phenotypes in rmCIRP‐treated ICAM‐1+ BMDN were significantly higher than those in ICAM‐1− BMDN. Following sepsis the ICAM‐1+ neutrophils in the lungs showed significantly higher levels of iNOS and NETs compared to ICAM‐1− neutrophils. We further revealed that ICAM‐1 and NETs were co‐localized in the neutrophils treated with rmCIRP. CIRP−/− mice showed significant improvement in their survival outcome (78% survival) over that of WT mice (48% survival) in sepsis. Thus, CIRP could be a novel therapeutic target for regulating iNOS producing and NETs forming ICAM‐1+ neutrophils in the lungs during sepsis. [ABSTRACT FROM AUTHOR]

Therapeutic hypothermia is well known for its protective effect against brain injury after cardiac arrest, but the exact mechanism remains unclear. Cold-inducible RNA-binding protein (CIRP), a member of cold shock protein, enables mammalian cells to withstand decreased temperature by regulating gene translation. However, the role of CIRP in global cerebral ischemia after therapeutic hypothermia has not been clearly elucidated. In the present study, rats resuscitated from 4 min of cardiac arrest were separately treated with therapeutic hypothermia (immediately after return of spontaneous circulation (ROSC); targeted temperature at 33 °C) and therapeutic normothermia (targeted temperature at 36.8 °C) for 6 h. The hippocampus was harvested at 0 h (baseline), 6 h, 12 h, 1 day, 3 days, and 7 days after ROSC. The expression of CIRP messenger RNA (mRNA) was assessed by real-time PCR. CIRP and mitochondrial apoptosis-associated proteins were tested by Western blot. The histological changes and neurological function were respectively evaluated by hematoxylin-eosin staining and neurological deficit score (NDS). Compared with baseline, rats resuscitated from cardiac arrest showed increased expression of CIRP, Bax, Caspase 9, and Caspase 3 and decreased expression of Bcl-2 in hippocampus ( P < 0.05). However, therapeutic hypothermia after ROSC alleviated the alterations of Bax, Caspase 9, Caspase 3, and Bcl-2, while further increased the hippocampal expression of CIRP mRNA and protein, when compared with the normothermia rats ( P < 0.05). In addition, compared with the therapeutic normothermia rats, histopathological damage in CA1 zone and NDS were respectively decreased and increased in the hypothermia rats ( P < 0.05). Our findings suggest that 32 °C therapeutic hypothermia exerts an important neuroprotective effects by up-regulating CIRP expression and inhibiting mitochondrial apoptosis factor production in the cardiac arrest rat model. [ABSTRACT FROM AUTHOR]

Phosphorus is a macro nutrients and it becomes the main limiting factor for maize growth on Ultisols. Rock phosphate is one of P fertilizer sources contains high P and Ca content, slow release and soluble under acid condition, suitable for acid soil. The objective of this research is to study the solubility of rock phosphate and its influence to the growth and yield of maize on Ultisols. This research was conducted in the research laboratory and the green house of Indonesia Soil Research Institute (ISRI). Ultisols soil samples were taken from oil palm plantation in North Sumatera. Christmas Island Rock Phosphate (CIRP) and Tunisia rock phosphate was used in this research. There are two activity conducted simultaneously, which is application of rock phosphate on Ultisols under plant indicator and without plant. The experimental design is randomized complete block design with 5 treatments and 3 replications. The pot experiment without plant was conducted using1 kg of soil sample mixed with rock phosphate treatment and placed into plastic plot. During 3 months of incubation, soil water content was maintained under field capacity by aquadest. The dosages of P fertilizer were 0, 157.6 and 315.2 kg P2O5 ha-1. Soil samples were taken on 1, 2, and 3 months after P fertilization for measuring pH, P content (by HCl 25 %, Bray 1 and Olsen extraction), P-inorganic fractionation (Al-P, Fe-P, Rs-P and Ca-P), exchangeable acidity (Al and H), Al and free of Fe oxide. The pot experiments with maize were conducted using 7.5 kg soil samples mixed with 2 different rock phosphates as a treatment. The dosages of rock phosphate were similar with experiment 1. Urea 300 kg and 150 kg KCl ha1 were applied as a basal fertilizer. Maize was planted two seed per pot and maintain until harvest time. The result of the research shows that soil P content increase with RP application, Tunisia rock phosphate give higher response than the CIRP. Direct application of rock phosphate with Fe and Al content did not increase significantly the Aldd, Al2O3 and Fe2O3 content on soil. Application of CIRP and Tunisia rock phosphate increased maize growth and yield significantly.

RNA binding proteins (RBPs) regulate gene expression by controlling mRNA export, translation, and stability. When altered, some RBPs allow cancer cells to grow, survive, and metastasize. Cold-inducible RNA binding protein (CIRP) is overexpressed in a subset of breast cancers, induces proliferation in breast cancer cell lines, and inhibits apoptosis. Although studies have begun to examine the role of CIRP in breast and other cancers, its role in normal breast development has not been assessed. We generated a transgenic mouse model overexpressing human CIRP in the mammary epithelium to ask if it plays a role in mammary gland development. Effects of CIRP overexpression on mammary gland morphology, cell proliferation, and apoptosis were studied from puberty through pregnancy, lactation and weaning. There were no gross effects on mammary gland morphology as shown by whole mounts. Immunohistochemistry for the proliferation marker Ki67 showed decreased proliferation during the lactational switch (the transition from pregnancy to lactation) in mammary glands from CIRP transgenic mice. Two markers of apoptosis showed that the transgene did not affect apoptosis during mammary gland involution. These results suggest a potential in vivo function in suppressing proliferation during a specific developmental transition. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate whether developmental competence of mature vitrified–warmed yak ( Bos grunniens ) oocytes can be enhanced by supplemented insulin-like growth factor I (IGF-1) during in vitro maturation (IVM), and its relationship with the expression of cold-inducible RNA-binding protein (CIRP). In experiment 1, immature yak oocytes were divided into four groups, and IVM supplemented with 0, 50, 100 and 200 ng/mL IGF-1 was evaluated; the mRNA and protein expression levels of CIRP in mature oocytes in the four groups were evaluated using quantitative real-time PCR and western blotting analyses. In experiment 2, the mature yak oocytes in the four groups were cryopreserved using the Cryotop (CT) method, followed by chemical activation and in vitro culture for two days and eight days to determine cleavage, blastocyst rates, and total cell number in the blastocysts. Mature yak oocytes without vitrification served as a control group. The outcomes were as following: (1) the expression of CIRP in the matured oocytes was up-regulated in the IGF-1 groups and was highest expression was observed in the 100 ng/mL IGF-1 treatment group. (2) In the vitrified–warmed groups, the rates of cleavage and blastocyst were also highest in the 100 ng/mL IGF-1 treatment group (81.04 ± 1.06%% and 32.16 ± 1.01%), which were close to the rates observed in groups without vitrification (83.25 ± 0.85% and 32.54 ± 0.34%). The rates of cleavage and blastocyst in the other vitrified–warmed groups were 70.92 ± 1.32% and 27.33 ± 1.31% (0 ng/mL); 72.73 ± 0.74% and 29.41 ± 0.84% (50 ng/mL); 72.43 ± 0.61% and 27.61 ± 0.59% (200 ng/mL), respectively. There was no significant difference in the total cell number per blastocysts between the vitrified–warmed groups and group without vitrification. Thus, we conclude that the enhancement in developmental competence of mature yak vitrified–warmed oocytes after the addition of IGF-1 during IVM might result from the regulation of CIRP expression in mature yak oocytes prior to vitrification. [ABSTRACT FROM AUTHOR]