TNF-α-positive patients with recurrent pregnancy loss: The etiology and management.

BACKGROUND: Elevated levels of tumor necrosis factor-alpha (TNF-α) have been associated with adverse pregnancy outcomes, specifically recurrent pregnancy loss (RPL). These elevated levels may be associated with the presence of autoantibodies. Although TNF-α inhibitors have shown promise in improving pregnancy rates, further research is needed to comprehend their impact and mechanisms in RPL patients. OBJECTIVE: This study aims to investigate the association between elevated TNF-α levels and autoantibodies in RPL patients, as well as evaluate the effect of TNF-α inhibition on pregnancy outcomes. METHODS: A total of 249 RPL patients were included in this study. Serum levels of TNF-α, autoantibodies, and complement were measured and monitored. Among these patients, 138 tested positive for TNF-α, while 111 tested negative. The medical records of these patients were retrospectively evaluated. Additionally, 102 patients with elevated TNF-α levels were treated with TNF-α inhibitors, and their pregnancy outcomes were assessed. RESULTS: TNF-α-positive RPL patients had higher levels of complement C1q, anti-cardiolipin (ACL)-IgA, ACL-IgM ,ACL-IgG, thyroglobulin antibody, and Anti-phosphatidylserine/prothrombin IgM antibody, as well as a higher positive rate of antinuclear antibodies compared to TNF-α-negative patients (23.19% vs. 12.6%, P< 0.05). Conversely, complement C3 were lower in TNF-α-positive patients (t test, P< 0.05). The use of TNF-α inhibitors led to a reduction in the early abortion rate (13.7% vs. 44.4%, P< 0.001) and an improvement in term delivery rate (52.0% vs. 27.8%, P= 0.012). Furthermore, patients who used TNF-α inhibitors before 5 weeks of pregnancy had a lower early abortion rate (7.7% vs. 24.3%, P= 0.033) and a higher term delivery rate (69.2% vs. 48.6%, P= 0.033). CONCLUSION: TNF-α plays a role in the occurrence and development of RPL, and its expression is closely associated with autoantibodies and complements. TNF-α inhibitors increase the term delivery rate in TNF-α-positive RPL patients, and their use before 5 weeks of pregnancy may more beneficial.

Transcriptional Repressor BCL11A in Erythroid Cells.

BCL11A, a zinc finger repressor, is a stage-specific transcription factor that controls the switch from fetal (HbF, α2γ2) to adult (HbA, α2ß2) hemoglobin in erythroid cells. While BCL11A was known as a factor critical for B-lymphoid cell development, its relationship to erythroid cells and HbF arose through genome-wide association studies (GWAS). Subsequent work validated its role as a silencer of γ-globin gene expression in cultured cells and mice. Erythroid-specific loss of BCL11A rescues the phenotype of engineered sickle cell disease (SCD) mice, thereby suggesting that downregulation of BCL11A expression might be beneficial in patients with SCD and ß-thalassemia. Common genetic variation in GWAS resides in an erythroid-specific enhancer within the BCL11A gene that is required for its own expression. CRISPR/Cas9 gene editing of the enhancer revealed a GATA-binding site that confers a large portion of its regulatory function. Disruption of the GATA site leads to robust HbF reactivation. Advancement of a guide RNA targeting the GATA-binding site in clinical trials has recently led to approval of first-in-man use of ex vivo CRISPR editing of hematopoietic stem/progenitor cells (HSPCs) as therapy of SCD and ß-thalassemia. Future challenges include expanding access and infrastructure for delivery of genetic therapy to eligible patients, reducing potential toxicity and costs, exploring prospects for in vivo targeting of hematopoietic stem cells (HSCs), and developing small molecule drugs that impair function of BCL11A protein as an alternative option.

Metabolic reprogramming induced by DCA enhances cisplatin sensitivity through increasing mitochondrial oxidative stress in cholangiocarcinoma.

Background: Cholangiocarcinoma has obvious primary multidrug resistance and is generally resistant to cisplatin and other chemotherapy drugs and high glycolytic levels may be associated with chemotherapy resistance of cholangiocarcinoma cells. Dichloroacetate (DCA) is a specific inhibitor of PDK, which can promote mitochondrial aerobic oxidation process by activating PDH. In the past few years, there have been an increasing number of studies supporting the action of DCA against cancer, which also provided evidence for targeting metabolism to enhance the efficacy of cholangiocarcinoma chemotherapy. Methods: Glucose uptake and lactic acid secretion were used to detect cell metabolism level. Cell apoptosis and cell cycle were detected to confirm cell fate induced by cisplatin combined with DCA. Mito-TEMPO was used to inhibit mtROS to explore the relationship between oxidative stress and cell cycle arrest induced by DCA under cisplatin stress. Finally, PCR array and autophagy inhibitor CQ were used to explore the potential protective mechanism under cell stress. Results: DCA changed the metabolic model from glycolysis to aerobic oxidation in cholangiocarcinoma cells under cisplatin stress. This metabolic reprogramming increased mitochondrial reactive oxygen species (mtROS) levels, which promoted cell cycle arrest, increased the expression of antioxidant genes and activated autophagy. Inhibition of autophagy further increased the synergistic effect of DCA and cisplatin. Conclusion: DCA increased cisplatin sensitivity in cholangiocarcinoma cells via increasing the mitochondria oxidative stress and cell growth inhibition. Synergistic effects of DCA and CQ were observed in cholangiocarcinoma cells, which further increased the cisplatin sensitivity via both metabolic reprogramming and inhibition of the stress response autophagy.

MUC1 induces the accumulation of Foxp3+ Treg cells in the tumor microenvironment to promote the growth and metastasis of cholangiocarcinoma through the EGFR/PI3K/Akt signaling pathway.

Tumor microenvironment (TME) plays an important role in the progression of cholangiocarcinoma. This study aims to explore whether Mucin 1 (MUC1) regulates Foxp3+ Treg cells in the TME of cholangiocarcinoma through the epidermal growth factor receptor (EGFR)/phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. High-throughput sequencing dataset in the GEO database combined with GeneCards and Phenolyzer databases was used to obtain key genes in cholangiocarcinoma, followed by downstream pathway prediction. The relationship among MUC1, EGFR, and PI3K/Akt signaling pathway was explored. CD4+ T cells extracted from peripheral blood were induced to differentiate into Treg cells, followed by co-culture with cholangiocarcinoma cells. A mouse model was constructed to detect the role of MUC1 in the accumulation of Foxp3+ Treg cells, malignant phenotypes of cholangiocarcinoma, and tumorigenesis in vivo. MUC1, highly expressed in cholangiocarcinoma, might be involved in cholangiocarcinoma development. MUC1 interacted with the EGFR to activate the EGFR/PI3K/Akt signaling pathway. MUC1 overexpression could activate the EGFR/PI3K/Akt signaling pathway, which promoted the accumulation of Foxp3+ Treg cells in the TME and the malignant phenotypes of cholangiocarcinoma cells both in vitro and in vivo and enhanced tumorigenesis in vivo. MUC1 may interact with EGFR to activate the EGFR/PI3K/Akt signaling pathway, which induces the accumulation of Foxp3+ Treg cells, enhancing the malignant phenotypes of cholangiocarcinoma cells and tumorigenesis in vivo and ultimately augmenting cholangiocarcinoma growth and metastasis.

Advances in Renal Cell Carcinoma Drug Resistance Models.

Renal cell carcinoma (RCC) is the most common form of kidney cancer. Systemic therapy is the preferred method to eliminate residual cancer cells after surgery and prolong the survival of patients with inoperable RCC. A variety of molecular targeted and immunological therapies have been developed to improve the survival rate and prognosis of RCC patients based on their chemotherapy-resistant properties. However, owing to tumor heterogeneity and drug resistance, targeted and immunological therapies lack complete and durable anti-tumor responses; therefore, understanding the mechanisms of systemic therapy resistance and improving clinical curative effects in the treatment of RCC remain challenging. In vitro models with traditional RCC cell lines or primary cell culture, as well as in vivo models with cell or patient-derived xenografts, are used to explore the drug resistance mechanisms of RCC and screen new targeted therapeutic drugs. Here, we review the established methods and applications of in vivo and in vitro RCC drug resistance models, with the aim of improving our understanding of its resistance mechanisms, increasing the efficacy of combination medications, and providing a theoretical foundation for the development and application of new drugs, drug screening, and treatment guidelines for RCC patients.

Methylprednisolone alleviates multiple sclerosis by expanding myeloid-derived suppressor cells via glucocorticoid receptor ß and S100A8/9 up-regulation.

Methylprednisolone is an effective drug in the treatment of autoimmune disease, such as multiple sclerosis (MS), due to long-acting anti-inflammatory, antiallergic and immunosuppressant. Previous studies have noted the importance of myeloid-derived suppressor cells (MDSC) in MS progression. However, it is still not known whether methylprednisolone could influence the ratio and function of MDSC during MS treatment. In the current study, we found an increased ratio of MDSC at the onset of EAE in mice model; but methylprednisolone pulse therapy (MPPT) did not alter the percentage and suppressive function of MDSC during disease attenuation. However, the percentage of G-MDSC in PBMC significantly increased in patients with MS. Surprisingly, relapsing MS patients showed a significant increase in both M-MDSC and G-MDSC after MPPT. The disease remission positively correlated expansion of MDSC and expression of arginase-1. Additionally, MPPT reduced the expression of inhibitory glucocorticoid (GCs) receptor ß subunit on MDSC while elevating serum levels of immune regulatory S100A8/A9 heterodimer. Thus, MDSC dynamics and function in mouse EAE differ from those in human MS during MPPT. Our study suggested that GCs treatment may help relieve the acute phase of MS by expanding MDSC through up-regulating of GR signalling and S100A8/A9 heterodimers.

Biologically active 1,25-dihydroxyvitamin D3 protects against experimental sepsis by negatively regulating the Toll-like receptor 4/myeloid differentiation primary response gene 88/Toll-IL-1 resistance-domain-containing adapter-inducing interferon-ß signaling pathway.

The hormonally active form of vitamin D (VD), 1,25­dihydroxyvitamin D3, has been reported to be a key immunoregulator in the reduction of inflammation. In this study, we investigated the effects of VD in an experimental sepsis cell model, and the underlying mechanisms. The sepsis cell model was first established in monocytes, isolated from newborns and healthy adults, which were stimulation with lipopolysaccharide (LPS). We observed that cell viability was significantly impaired in the monocytes after LPS stimulation, using a Cell Counting Kit­8 and trypan blue assays. Additionally, ELISA revealed that LPS stimulation significantly elevated the expression of interleukin 6 (IL­6), IL­10 and tumor necrosis factor­α (TNF­α). The expression levels of Toll­like receptor (TLR4), myeloid differentiation primary response gene 88 (MyD88), and Toll­IL­1 resistance­domain­containing adapter­inducing interferon­ß (TRIF) mRNA were also significantly elevated under LPS stimulation using reverse transcription­quantitative PCR and western blot analysis. VD treatment could significantly suppress the effects of LPS simulation on monocytes by negatively regulating inflammatory cytokines and TLR4/MyD88/TRIF signaling. Furthermore, a regulatory feedback mechanism was proposed to involve TLR4, MyD88 and TRIF in the sepsis cell model. In conclusion, VD may effectively decrease the release of inflammatory cytokines by inhibiting the TLR4/MyD88/TRIF signaling pathway, could be considered as a potential therapeutic agent for the treatment of sepsis.

Association between indel polymorphism (rs145204276) in the promoter region of lncRNA GAS5 and the risk of febrile convulsion.

BACKGROUND: This study aimed to explore the regulatory relationship between growth arrest special 5 (GAS5) and interleukin-1ß (IL-1ß) implicated in the development of febrile seizure (FS). METHOD: The presence of FS and the genotype of GAS5 were used as two different indicators to divide the 50 newborn babies, recruited in this study, into different groups. The potential regulatory relationship among GAS5, miR-21, and IL-1ß was identified by measuring their expression using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry assays among different sample groups. Computational analyses and luciferase assays were also conducted to verify the interaction between GAS5, miR-21, and IL-1ß. RESULT: GAS5 and IL-1ß expression was upregulated in cells collected from FS patients or genotyped as INS/DEL and DEL/DEL, whereas the expression of miR-21 was decreased in above samples, indicating a negative relationship between miR-21 and GAS5/IL-1ß. Results of the computational analysis showed that miR-21 directly bound to and increased the expression of GAS5, whereas the expression of IL-1ß was suppressed by miR-21. In the presence of GAS5, the expression of miR-21 was lowered, whereas the expression of IL-1ß was increased. CONCLUSION: The results obtained in this study supported the conclusion that GAS5 negatively regulated the expression of miR-21, which in turn negatively regulated the expression IL-1ß. Therefore, the overexpression of GAS5 could decrease the magnitude of FS.

NAFLD Induction Delays Postoperative Liver Regeneration of ALPPS in Rats.

BACKGROUND: Associating liver partition and portal vein ligation (ALPPS) is a promising two-step hepatectomy that is beneficial for accumulative regeneration of the future liver remnant (FLR) and avoids postoperative liver failure. AIMS: Our study aimed to evaluate whether nonalcoholic fatty liver disease affected the liver regeneration induced by ALPPS. METHODS: Sprague-Dawley rats fed a high-fat diet were used to construct the NAFLD model. ALPPS were performed, and blood and future liver remnant samples were collected at postoperative days 1 (POD1), POD3, and POD7. RESULTS: The hepatic regeneration rate (HRR) of ALPPS was higher than that of portal vein ligation (PVL) at POD3 and POD7 (p < 0.05), and the number of Ki-67-positive hepatocytes (POD3) and CD68-positive Kupffer cells (POD7) per visual field was higher in the ALPPS group than in the PVL group (p < 0.05). The serum TNF-α, hepatocyte growth factor protein, and the serum IL-6 level were higher in the ALPPS group than in the PVL group at POD3 and POD7. Compared with those of the standard laboratory diet (SLD)-fed rats, the rats with NAFLD exhibited a decrease in the HRR, Ki-67-positive hepatocytes, and CD68-positive Kupffer cells in the FLR. The number of CD68-positive Kupffer cells was lower in rats with NAFLD than that in SLD-fed rats; noteworthily, the serum level of IL-6 and TNF-α changed dramatically after surgeries. CONCLUSIONS: NAFLD induction delayed liver regeneration induced by the ALPPS procedure, which might be associated with hepatocyte proliferation and the number of Kupffer cells.

Mutations in GAS5 affect the transformation from benign prostate proliferation to aggressive prostate cancer by affecting the transcription efficiency of GAS5.

BACKGROUND: In this study, we aimed to explore the effects of GAS5 single-nucleotide polymorphisms (SNPs) on GAS5 expression. And the signaling pathways underlying the function of GAS5 during the pathogenesis of prostate cancer (PC) were clarified. MATERIALS AND METHODS: Patients with PC were recruited and grouped according to their specific genotypes of rs55829688 and rs145204276. Kaplan-Meier overall survival curves were calculated and compared among different groups. Real-time polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry (IHC) assays were conducted to examine the expression of different factors involved in PC. And computational analyses and luciferase assays were conducted to clarify the regulatory relationships among the above factors. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), flow cytometry, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays were used to evaluate cell viability and apoptosis. RESULTS: The expression of GAS5, PDCD4, PTEN, and AKT was decreased gradually in the order of patient Group 1-4, whereas the expression of microRNA-21 (miR-21) and miR-1284 showed an opposite trend. GAS5 was identified to target miR-21 and miR-1284, whereas miR-21 and miR-1284 regulated the expression of PDCD4/PTEN and AKT, respectively. Moreover, the GAS5/miR-21/PDCD4/PTEN and GAS5/miR-1284/AKT signaling pathway was found to be closely related to the tumorigenesis of PC. CONCLUSIONS: GAS5 SNPs affected the survival rate and prognosis in patients with PC via regulating the expression of miR-21/miR-1284, which in turn affected the expression of PDCD4, PTEN, and AKT. GAS5 downregulated the expression of miR-21/miR-1284, thus leading to the elevated expression of key regulators of apoptosis. Therefore, the GAS5 SNPs may be used as key indicators for the diagnosis and prognosis prediction of PC.

Differentiation expression of toll-like receptor4 (TLR4) caused by the dysregulation of microRNA-140-5p is responsible for the development of postoperation infection.

BACKGROUND: Toll-like receptor4 (TLR4) has proven to be an important factor that's responsible for the development of postoperation infection. MicroRNAs (miRNAs) are widely regarded as key mediators of gene expression. The objectives of our study were to identify miRNA(s) and the target genes differentially expressed in monocytes in the individuals with postoperation infection. METHODS: MiRNA microarrays were performed to identify and compare miRNA expression in monocytes from those with or without postoperative infection. In-silico analysis was used to further investigate the target miRNAs and finally, luciferase assay and real-time polymerase chain reaction (PCR) were performed to confirm the target miRNA identified. Enzyme-linked immunosorbent assay, real-time PCR and Western-blot were performed to explore the role of miR-140 involved in postoperation infection. RESULTS: MiRNA microarray results showed that ten miRNAs were upregulated in the postoperation infection group, while six miRNAs were downregulated, compared with those in the postoperation group without infection. Computational analysis was further performed to reveal that four miRNAs (miR-140, miR-7, miR-448, and miR-217) targeted the 3'-untranslated region (UTR) of TLR4 mRNA. The luciferase assay showed that only miR-140 inhibited luciferase activity of wild-type TLR4 3'-UTR and the luciferase activity of the cells cotransfected with miR-7, miR-448 or miR-217 and wild-type or mutant TLR4 3'-UTR was comparable with the control. Furthermore, only miR-140 levels were significantly lower in the postoperation infection group, while levels of miR-217, miR-7, and miR-448 showed no obvious difference between the postoperation infection and postoperation without infection groups. TLR4, tumor necrosis factor-α (TNF-α), and IL-6 levels were much higher in the postoperation infection group. In comparison with the control group, TLR4, TNF-α and Interleukin 6 (IL-6) levels in cells were decreased following transfection with miR-140 mimics and TLR4 small interfering RNA. However, the cells treated with lipopolysaccharides increased TLR4, TNF-α, and IL-6 levels. CONCLUSION: This study demonstrates that miR-140 is differentially expressed in monocytes collected from patients diagnosed with postoperation infection. The downregulation of miR-140 cause upregulation of toll-like receptor4 (TLR4), a proinflammatory factor, and is associated with infection risk in patients received surgery.

Effect of sitagliptin, a DPP-4 inhibitor, against DENA-induced liver cancer in rats mediated via NF-κB activation and inflammatory cytokines.

The target of the current research was to investigate the anticancer activity of sitagliptin on diethylnitrosamine (DENA)-induced cancer in the liver. Wistar rats were treated with or without sitagliptin before DENA treatment. We detected liver weight, blood glucose, and histopathology of the liver. Serum biochemical markers like serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), serum alkaline phosphatase (SALP), gamma-glutamyl transpeptidase (GGTP), total bilirubin (TBR), total protein (TPR), and albumin (ALB) were also evaluated. In addition, lipid profile parameters comprising total cholesterol (TC), triglycerides, and high-density lipoprotein were also measured. Inflammatory mediators like interleukin-6 (IL-6), interleukin-1beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α) were determined in liver homogenate. Furthermore, the activity of nuclear factor (NF-κB) was also measured. Our results showed that sitagliptin (10 and 20 mg/kg) in a dose-dependent manner expressively decreased the DENA-induced elevation of SGPT, SGOT, SALP, and GGTP. Whereas sitagliptin (10 and 20 mg/kg) in a dose-dependent mode reduced the level of TBR and increased the TPR and ALB as well as improved the liver histopathology alterations in DENA-exposed rats. Lipid profile was also restored by the sitagliptin (10 and 20 mg/kg) in a DENA-treated rats. The level of IL-1ß, IL-6, and TNF-α were suggestively suppressed. Moreover, pretreatment with sitagliptin (10 and 20 mg/kg) prevented the activation of NF-κB. In conclusion, sitagliptin (10 and 20 mg/kg) has a potential protective effect against DENA-induced liver cancer by inhibition of inflammation and NF-κB activation.

Effects of vitamin D on apoptosis of T-lymphocyte subsets in neonatal sepsis.

Effect of vitamin D on apoptosis of peripheral blood T-lymphocyte subsets in treatment of neonatal sepsis was investigated. A total of 150 neonatal patients with sepsis were randomly divided into vitamin D treatment group (observation group) and treatment control group, while 100 healthy newborns were selected as healthy control group. T-lymphocyte subsets were detected by flow cytometer, the levels of tumor necrosis factor-α, interleukin-1 and calcitonin were determined by double-antibody immunoluminometric assay, and the effect of vitamin D on the above indicators in the treatment of sepsis was observed. Serum 25(OH)D (22.52±5.56 mg/l) in the treatment group was obviously increased compared with that in the treatment group (14.85±6.14 mg/l) (P<0.05), but the levels in the two groups were remarkably lower than that in the normal control group (26.38±6.56 mg/l), and the differences were statistically significant (P<0.05). Cluster of differentiation 4 (CD4+) T-lymphocyte subset in sepsis patients was obviously reduced compared with that in the healthy control group (P<0.01); the difference in comparison of CD8+ T-lymphocyte subset between sepsis patients and healthy people was not statistically significant (P>0.05). After treatment for 72 h, CD4+ T-lymphocytes were increased, and the ratio of CD4+ to CD8+ was close to 1, suggesting that the effect was superior to that in the treatment control group. The inflammatory factor levels in children with sepsis were evidently higher than those in the healthy control group (P<0.01), and high-level states of inflammatory factors were significantly improved after treatment with vitamin D for 72 h, indicating that the effect was superior to that in the treatment group. The results indicated that the prognosis of sepsis patients treated with vitamin D is improved, and the mechanism may be achieved by regulating T-lymphocyte subsets and inflammatory factors.

Humanized mice reveal an essential role for human hepatocytes in the development of the liver immune system.

The liver is an immunological organ with a distinct immune cell profile. Although the composition and function of liver immune cells have been widely investigated, the mechanisms regulating the development and homeostasis of the specialized immune system, especially in humans, remain largely unknown. Herein, we address this question in humanized mice (hu-mice) that were constructed by transplantation of human fetal thymus and CD34+ hematopoietic stem/progenitor cells in immunodeficient mice with or without autologous human hepatocyte engraftment. Although the levels of human immune cell reconstitution in peripheral blood and spleen were comparable between hu-mice with and without human hepatocyte engraftment, the former group showed that human immune cell reconstitution in the liver was significantly improved. Notably, human immune cells, including Kupffer cells, dendritic cells and natural killer cells, were shown to be closely colocalized with human hepatocytes in the liver. Human hepatocytes engrafted in the mouse liver were found to produce IL-3, IL-15, GM-CSF, M-CSF, MCP-1, CXCL-1 and CXCL-10, which are known to be important for immune cell development, differentiation, tissue migration and retention, and have no or poor cross-reaction between humans and mice. Furthermore, human hepatocytes were able to support human immune cell survival and expansion in an in vitro co-culture assay. This study demonstrates an essential role for hepatocytes in the development and maintenance of the liver immune cell profile. The hu-mouse model with human autologous immune cell and hepatocyte reconstitution has potential for use in studies of the pathogenesis of liver immune disorders such as hepatotropic virus infections.

Cyclin A Turns on Bora to Light the Path to Mitosis.

Mitotic entry is an irreversible cell-cycle transition that requires the robust, rapid activation of cyclin-dependent kinase 1 (Cdk1). In this issue of Developmental Cell, Vigneron et al. (2018) identify cyclin A-Cdk1 as the factor triggering mitotic entry through the phosphorylation of Bora, an activator of the kinase Aurora A.

MicroRNA-135a is up-regulated and aggravates myocardial depression in sepsis via regulating p38 MAPK/NF-κB pathway.

MicroRNA-135a (miR-135a) is implicated in the pathological processes of several cancers. However, the roles and regulatory mechanism of miR-135a in sepsis-induced myocardial depression (MD) remain largely unknown. In this study, the serum of patients with sepsis and healthy controls was obtained. The miR-135a expression was then measured. Then lentiviruses (miR-135a mimic, inhibitor and scramble control) were transfected into BALB/c mice. After 4days of transfection, polymicrobial sepsis model was established by cecal ligation and puncture (CLP) surgery. The serum tumor-necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 were detected. Cardiac function was assessed. In addition, the protein expressions of p38 MAPK/NF-κB pathway-related proteins were determined. Besides, SB203580 and JSH-23, the inhibitors of p38 MAPK and NF-κB respectively, were used to treat the isolated ventricular myocytes in vitro. MiR-135a was significantly up-regulated in the serum of patients with sepsis. In comparison with CLP group, the concentrations of TNF-α, IL-1ß and IL-6 and the expressions of p-p38 and p-p65 in CLP+miR-135a mimic group were significantly increased, while markedly decreased in CLP+miR-135a inhibitor group. Moreover, EF, FS, LVdP/dt (max), LVdP/dt (min) and LVDP of CLP+miR-135a mimic group were all significantly decreased, while markedly increased in CLP+miR-135a inhibitor group. Besides, the increased expressions of p-p38 and p-p65, decreased expression of p-IKBα and the decreased percentage of contraction amplitude in miR-135a mimic group were markedly reversed by SB203580 or JSH-23 treatments. Up-regulation of miR-135a could aggravate sepsis-induced inflammation and myocardial dysfunction via activation of p38 MAPK/NF-κB pathway.

Biochemical and Functional Assays of Human Cohesin-Releasing Factor Wapl.

During the cell cycle, duplicated sister chromatids become physically connected during S phase through a process called sister-chromatid cohesion. Cohesion is terminated during the metaphase-to-anaphase transition to trigger sister-chromatid segregation. The establishment and dissolution of cohesion are highly regulated by the cohesin complex and its multitude of regulators. In particular, the cohesin regulator Wapl promotes the release of cohesin from chromosomes during both interphase and mitosis. Here, we describe in vitro protein binding assays between Wapl and a cohesin subcomplex, and cellular assays in human cells that probe the functions of Wapl in cohesin release.

Structural Basis and IP6 Requirement for Pds5-Dependent Cohesin Dynamics.

The ring-shaped cohesin complex regulates transcription, DNA repair, and chromosome segregation by dynamically entrapping chromosomes to promote chromosome compaction and sister-chromatid cohesion. The cohesin ring needs to open and close to allow its loading to and release from chromosomes. Cohesin dynamics are controlled by the releasing factors Pds5 and Wapl and the cohesin stabilizer Sororin. Here, we report the crystal structure of human Pds5B bound to a conserved peptide motif found in both Wapl and Sororin. Our structure establishes the basis for how Wapl and Sororin antagonistically influence cohesin dynamics. The structure further reveals that Pds5 can bind inositol hexakisphosphate (IP6). The IP6-binding segment of Pds5B is shaped like the jaw of a plier lever and inhibits the binding of Scc1 to Smc3. We propose that Pds5 stabilizes a transient, open state of cohesin to promote its release from chromosomes.

Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion.

Orderly termination of sister-chromatid cohesion during mitosis is critical for accurate chromosome segregation. During prophase, mitotic kinases phosphorylate cohesin and its protector sororin, triggering Wapl-dependent cohesin release from chromosome arms. The shugoshin (Sgo1)-PP2A complex protects centromeric cohesin until its cleavage by separase at anaphase onset. Here, we report the crystal structure of a human cohesin subcomplex comprising SA2 and Scc1. Multiple HEAT repeats of SA2 form a dragon-shaped structure. Scc1 makes extensive contacts with SA2, with one binding hotspot. Sgo1 and Wapl compete for binding to a conserved site on SA2-Scc1. At this site, mutations of SA2 residues that disrupt Wapl binding bypass the Sgo1 requirement in cohesion protection. Thus, in addition to recruiting PP2A to dephosphorylate cohesin and sororin, Sgo1 physically shields cohesin from Wapl. This unexpected, direct antagonism between Sgo1 and Wapl augments centromeric cohesion protection.

Structure of the human cohesin inhibitor Wapl.

Cohesin, along with positive regulators, establishes sister-chromatid cohesion by forming a ring to circle chromatin. The wings apart-like protein (Wapl) is a key negative regulator of cohesin and forms a complex with precocious dissociation of sisters protein 5 (Pds5) to promote cohesin release from chromatin. Here we report the crystal structure and functional characterization of human Wapl. Wapl contains a flexible, variable N-terminal region (Wapl-N) and a conserved C-terminal domain (Wapl-C) consisting of eight HEAT (Huntingtin, Elongation factor 3, A subunit, and target of rapamycin) repeats. Wapl-C folds into an elongated structure with two lobes. Structure-based mutagenesis maps the functional surface of Wapl-C to two distinct patches (I and II) on the N lobe and a localized patch (III) on the C lobe. Mutating critical patch I residues weaken Wapl binding to cohesin and diminish sister-chromatid resolution and cohesin release from mitotic chromosomes in human cells and Xenopus egg extracts. Surprisingly, patch III on the C lobe does not contribute to Wapl binding to cohesin or its known regulators. Although patch I mutations reduce Wapl binding to intact cohesin, they do not affect Wapl-Pds5 binding to the cohesin subcomplex of sister chromatid cohesion protein 1 (Scc1) and stromal antigen 2 (SA2) in vitro, which is instead mediated by Wapl-N. Thus, Wapl-N forms extensive interactions with Pds5 and Scc1-SA2. Wapl-C interacts with other cohesin subunits and possibly unknown effectors to trigger cohesin release from chromatin.