Elevated cell-free fetal DNA contributes to placental inflammation and antiangiogenesis via AIM2 and IFI16 during pre-eclampsia

Accumulated evidence has shown that pre-eclampsia (PE) is related to both maternal and utero-placental antiangiogenesis and inflammation. Remarkably, an elevated cell-free fetal DNA (cffDNA) level has been found in maternal circulation; however, it remains unclear whether this DNA can induce activation of cytosolic DNA sensor signaling pathways and lead to the development of PE. In this study, we found that trophoblast cells constitutively expressed the cytosolic DNA sensors, absent in melanoma 2 (AIM2) and interferon-inducible protein 16 (IFI16). The cffDNA and pro-inflammatory and antiangiogenic factors were present at higher concentrations in PE compared with the control group and correlated with the severity of PE. DNA stimulation significantly increased the AIM2 and IFI16 levels, consistent with the elevated AIM2 and IFI16 expression in women with PE, and elicited increased production of AIM2-mediated interleukin IL-8 (IL-8), IL-6 and CC chemokine ligand 2 (CCL2) and IFI16-mediated sEndoglin, sFlt-1 and CXCL10. Furthermore, enhancement of the inflammatory response was found to be induced by DNA exposure, but DNA exposure did not induce PE-like symptoms in pregnant mice. It is possible that elevated cffDNA could reflect the degree of placental damage and trigger cytosolic DNA sensor activation, which disrupts the immunity balance and, consequently, contributes to inflammatory and antiangiogenic responses. In conclusion, the results of this study suggest that circulating cffDNA levels are increased in preeclamptic women and act through AIM2 and IFI16 activation to promote the production of pro-inflammatory and antiangiogenic factors, which correlate with the severity of the disease, and may offer insights into the etiology and pathogenesis of PE.     

Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process

Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3′-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3′-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3′-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences.     

Inhibition of ROS-Activated p38MAPK Pathway is Involved in the Protective Effect of H2S Against Chemical Hypoxia-Induced Inflammation in PC12 Cells

We have demonstrated the neuroprotection of hydrogen sulfide (H₂S) against chemical hypoxia-induced injury by inhibiting p38MAPK pathway. The present study attempts to evaluate the effect of H₂S on chemical hypoxia-induced inflammation responses and its mechanisms in PC12 cells. We found that treatment of PC12 cells with cobalt chloride (CoCl₂, a hypoxia mimetic agent) enhanced IL-6 secretion, nitric oxide (NO) generation and expression levels of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). L-canavanine, a selective iNOS inhibitor, partly blocked CoCl₂-induced cytotoxicity, apoptosis and mitochondrial insult. In addition, 7-Nitroindazole (7-NI), an inhibitor of nNOS, also partly attenuated the CoCl₂-induced cytotoxicity. The inhibition of p38MAPK by SB203580 (a selective p38MAPK inhibitor) or genetic silencing of p38MAPK by RNAi (Si-p38) depressed not only CoCl₂-induced iNOS expression, NO production, but also IL-6 secretion. In addition, N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger, conferred a similar protective effect of SB203580 or Si-p38 against CoCl₂-induced inflammatory responses. Importantly, pretreatment of PC12 cells with exogenous application of sodium hydrosulfide (a H₂S donor, 400 μmol/l) for 30 min before exposure to CoCl₂ markedly attenuated chemical hypoxia-stimulated iNOS and nNOS expression, NO generation and IL-6 secretion as well as p38MAPK phosphorylation in PC12 cells. Taken together, we demonstrated that p38MAPK-iNOS pathway contributes to chemical hypoxia-induced inflammation and that H₂S produces an anti-inflammatory effect in chemical hypoxia-stimulated PC12 cells, which may be partly due to inhibition of ROS-activated p38MAPK-iNOS pathway.     

Nicotine-Induced Neuroprotection Against Ischemic Injury Involves Activation of Endocannabinoid System in Rats

Nicotine has been reported to exert certain protective effect in the Parkinson’s and Alzheimer’s diseases. Whether it has a similar action in focal cerebral ischemia was unclear. In the present study, rats received either an injection of (?)-nicotine hydrogen tartrate salt (1.2 mg/kg, i.p.) or the vehicle 2 h before the 120 min middle cerebral artery occlusion. Neurological deficits and histological injury were assessed at 24 h after reperfusion. The content of endocannabinoids and the expression of cannabinoid receptor CB1 in brain tissues were determined at different time points after nicotine administration. Results showed that nicotine administration ameliorated neurological deficits and reduced infarct volume induced by cerebral ischemia in the rats. The neuroprotective effect was partially reversed by CB1 blockage. The content of the endocannabinoids N-arachidonylethanolamine and 2-arachidonoylglycerol, as well as the expression of cannabinoid receptor CB1 were up-regulated in brain tissues after nicotine delivery. These results suggest that endogenous cannabinoid system is involved in the nicotine-induced neuroprotection against transient focal cerebral ischemia.     

Effects of Sevoflurane on Self-Renewal Capacity and Differentiation of Cultured Neural Stem Cells

Sevoflurane anesthesia in infant rats can result in long-term cognitive impairment, possibly by inhibiting neurogenesis. The hippocampus is critical for memory consolidation and is one of only two mammalian brain regions where neural stem cells (NSCs) are renewed continuously throughout life. To elucidate the pathogenesis of sevoflurane-induced cognitive dysfunction, we measured the effects of clinical sevoflurane doses on the survival, proliferation, and differentiation of hippocampal NSCs. Neural stem cells were isolated from Sprague–Dawley rat embryos, expanded in vitro, and exposed to sevoflurane at 0.5, 1, or 1.5 minimal alveolar concentration (MAC) for 1 or 6 h. Two days after treatment, cell viability, cytotoxicity, and apoptosis rate were estimated by WST-1 assay, lactate dehydrogenase (LDH) activity, and TdT-mediated dUTP-biotin nick end labeling (TUNEL), respectively, while proliferation rate was assessed by 5-ethynyl-2′-deoxyuridine (BrdU) incorporation and Ki67 staining. Differentiation was assayed 7 days after treatment by immunocytochemistry and Western blots of neuron and glial markers. The phosphorylation level of p44/42 extracellular regulated kinases (ERK1/2) was measured in the proliferation and differentiation phases respectively. Sevoflurane at 1 MAC or 1.5 MAC for 1 h increased viable cell number whereas a 6 h exposure at these same concentrations suppressed proliferation and promoted apoptotic death (P < 0.01). Sevoflurane had no effect on NSC differentiation, and a sub-clinical concentration (0.5 MAC) altered neither proliferation nor viability. The phosphorylation level of ERK1/2 increased after 1 h of 1 MAC or 1.5 MAC of sevoflurane exposure in the proliferation phase, but not in the differentiation phase. Brief (1 h) exposure to sevoflurane at clinical concentrations enhanced proliferation of cultured NSCs possibly mediated by ERK1/2, but a 6 h exposure suppressed proliferation and induced apoptosis. Prolonged sevoflurane exposure may decrease the self-renewal capacity of hippocampal NSCs, resulting in cognitive deficits.     

Novel SLC20A2 mutations identified in southern Chinese patients with idiopathic basal ganglia calcification

Idiopathic basal ganglia calcification (IBGC) is a rare neuropsychiatric disorder characterized by bilateral and symmetric cerebral calcifications. Recently, SLC20A2 was identified as a causative gene for familial IBGC, and three mutations were reported in a northern Chinese population. Here, we aimed to explore the mutation spectrum of SLC20A2 in a southern Chinese population. Sanger sequencing was employed to screen mutations within SLC20A2 in two IBGC families and 14 sporadic IBGC cases from a southern Han Chinese population. Four novel mutations (c.82G > A p.D28N, c.185T > C p.L62P, c.1470_1478delGCAGGTCCT p.Q491_L493del and c.935-1G > A) were identified in two families and two sporadic cases, respectively; none were detected in 200 unrelated controls. No mutation was found in the remaining 12 patients. Different mutations may result in varied phenotypes, including brain calcification and clinical manifestations. Our study supports the hypothesis that SLC20A2 is a causative gene of IBGC and expands the mutation spectrum of SLC20A2, which facilitates the understanding of the genotype–phenotype correlation of IBGC.     

Cloning,characterization and expression of a cDNA encoding a granulin-like polypeptide in Ciona savignyi

Previous study in our laboratory confirmed that a novel polypeptide, CS5931 derived from Ciona savignyi possesses potent antitumor activity. In the present study, the full length cDNA of CS5931 precursor, termed Cs-pgrn-1 was cloned. The complete cDNA sequence of this gene consists of 685 bp containing an open reading frame (ORF) of 522 bp (173 amino acid residues). In silico analysis revealed that the polypeptide consists of two identical domains, similar with granulin (GRN) found in other species, and each of the domain encodes a polypeptide identical with CS5931. Phylogenetic analysis confirmed that CS5931 shares high homology with Ciona intestinalis GRN and is conserved during evolution. The polypeptide also shows high similarity with human GRN A, B, and C. Prediction of 3D protein structure revealed the 3D structure of CS5931 is very similar with human GRN A. The CS5931 was expressed using a prokaryotic expression system and the purified polypeptide inhibited the growth of several tumor cell lines in vitro via apoptotic pathway. Our study revealed that CS5931 has the potential to be developed as a novel antitumor agent.     

FATP1 silence inhibits the differentiation and induces the apoptosis in chicken preadipocytes

FATP1 plays an important role in the trafficking of free fatty acids in adipocytes, however, its precise function and relationship with other fatty acid transporters all remain poorly understood. In this study, FATP1 gene silencing was induced by transfecting siRNA of target sequence into chicken preadipocytes, then the expression of FABP was found down-regulated while the expression of FAT was raised. In addition, differential inhibition of the cells was observed and the expressions of PPARγ and C/EBPα were found down-regulated. Moreover, the silencing also induced the down-regulation of FAS and inhibited the adipogenesis in adipocytes. Of specific interest here was that FATP1 silencing significantly improved the expressions and activities of cell apoptotic factors Caspases 3 and BCL2 associated X protein (Bax). Consequently, FATP1 deficiency prevented the differentiation while induced apoptosis in chicken preadipocytes.     

Immune responses induced in HHD mice by multiepitope HIV vaccine based on cryptic epitope modification

CD8+ T cells play an important role in early HIV infection. However, HIV has the capacity to avoid specific CTL responses due to a high rate of mutation under selection pressure. Although the HIV proteins, gag and pol, are relatively conserved, these sequences generate low-affinity MHC-associated epitopes that are poorly immunogenic. Here, we applied an approach that enhanced the immunogenicity of low-affinity HLA-A2.1-binding peptides. The first position with tyrosine (P1Y) substitution enhanced the affinity of HLA-A2.1-associated peptides without altering their antigenic specificity. More importantly, P1Y variants efficiently stimulated in vivo native peptide-specific CTL that also recognized the corresponding naturally processed epitope. The potential to generate CTL against any low-affinity HLA-A2.1-associated peptide provides us with the necessary technique for identification of virus cryptic epitopes for development of peptide-based immunotherapy. Therefore, identification and modification of the cryptic epitopes of gal and pol provides promising candidates for HIV immunotherapy dependent upon efficient presentation by virus cells. Furthermore, this may be a breakthrough that overcomes the obstacle of immune escape caused by high rates of mutation. In this study, bioinformatics analysis was used to predict six low-affinity cryptic HIV gag and pol epitopes presented by HLA-A*0201. A HIV compound multi-CTL epitope gene was constructed comprising the gene encoding the modified cryptic epitope and the HIV p24 antigen, which induced a strong CD8+ T cell immune response regardless of the mutation. This approach represents a novel strategy for the development of safe and effective HIV prophylactic and therapeutic vaccines.     

Simvastatin inhibits the additive activation of ERK1/2 and proliferation of rat vascular smooth muscle cells induced by combined mechanical stress and oxLDL through LOX-1 pathway

Vein grafts interposed into arteries are susceptible to the development of atherosclerosis due to rapid increases in blood pressure. This process is accelerated in patients with hyperlipidemia. The molecular mechanism underlying this process is unknown. In this study, quiescent rat vascular smooth muscle cells (VSMCs) were treated in vitro with mechanical stretch stress (10% elongation) with and without oxLDL (25 μg/ml) in the presence and absence of simvastatin (2.5 μmol/L). The results demonstrate that stretch stress and oxLDL can each induce activation of ERK1/2 and Ki-67 expression in VSMCs, but the peak levels of ERK activation and Ki-67 expression were observed in groups subjected to both stretch stress and oxLDL. Simvastatin was found to inhibit increased ERK activation and Ki-67 expression in VSMCs subjected to stretch stress with or without oxLDL. Mechanically, simvastatin was also found to inhibit increased expression of LOX-1 (a receptor of oxLDL) in VSMCs subjected to stretch stress with or without oxLDL. Knockdown of LOX-1 via small interfering RNAs (siRNA-LOX-1) resulted in obvious inhibition of ERK activation in VSMCs subjected to stretch stress with and without oxLDL. These results suggest that combined stretch stress and oxLDL can additively promote the activation of ERK1/2 leading to accelerated proliferation of VSMCs (e.g. increased Ki-67 expression) via LOX-1 signal pathway. This was found to be partially inhibited by simvastatin. These results may provide important data for the treatment and prevention of hypertension with or without hyperlipidemia.