Calycosin inhibited MIF-mediated inflammatory chemotaxis of macrophages to ameliorate ischemia reperfusion-induced acute kidney injury.

BACKGROUND: Inflammatory macrophage infiltration plays a critical role in acute kidney disease induced by ischemia-reperfusion (IRI-AKI). Calycosin is a natural flavone with multiple bioactivities. This study aimed to investigate the therapeutic role of calycosin in IRI-AKI and its underlying mechanism. METHODS: The renoprotective and anti-inflammatory effects of calycosin were analyzed in C57BL/6 mice with IRI-AKI and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. RNA-seq was used for mechanism investigation. The molecular target of calycosin was screened by in silico methods and validated by surface plasmon resonance (SPR). Macrophage chemotaxis was analyzed using Transwell and agarose gel spot assays. RESULTS: Calycosin treatment significantly reduced serum creatinine and urea nitrogen and attenuated tubular destruction in IRI-AKI mice. Additionally, calycosin markedly suppressed NF-κB signaling activation and the expression of inflammatory mediators IL-1ß and TNF-α in IRI-AKI kidneys and LPS-stimulated RAW 264.7 cells. Interestingly, RNA-seq revealed calycosin remarkably downregulated chemotaxis-related pathways in RAW 264.7 cells. Among the differentially expressed genes, Ccl2/MCP-1, a critical chemokine mediating macrophage inflammatory chemotaxis, was downregulated in both LPS-stimulated RAW 264.7 cells and IRI-AKI kidneys. Consistently, calycosin treatment attenuated macrophage infiltration in the IRI-AKI kidneys. Importantly, in silico target prediction, molecular docking, and SPR assay demonstrated that calycosin directly binds to macrophage migration inhibitory factor (MIF). Functionally, calycosin abrogated MIF-stimulated NF-κB signaling activation and Ccl2 expression and MIF-mediated chemotaxis in RAW 264.7 cells. CONCLUSIONS: In summary, calycosin attenuates IRI-AKI by inhibiting MIF-mediated macrophage inflammatory chemotaxis, suggesting it could be a promising therapeutic agent for the treatment of IRI-AKI.

Calycosin pretreatment enhanced the therapeutic efficacy of mesenchymal stem cells to alleviate unilateral ureteral obstruction-induced renal fibrosis by inhibiting necroptosis.

Bone marrow-derived mesenchymal stem cells (MSCs) show antifibrotic activity in various chronic kidney diseases. Here, we aimed to investigate whether Calycosin (CA), a phytoestrogen, could enhance the antifibrotic activity of MSCs in primary tubular epithelial cells (PTECs) induced by TGF-ß1 and in a mouse model of unilateral ureteral obstruction (UUO). We found that MSCs treatment significantly inhibited fibrosis, and CA pretreatment enhanced the effects of MSCs on fibrosis in vitro. Consistent with the in vitro studies, MSCs alleviated tubular injury and renal fibrosis in mice after UUO, and CA-pretreated MSCs resulted in more significant improvements in tubular injury and renal fibrosis than MSCs after UUO. Moreover, MSCs treatment significantly inhibited necroptosis by repressing the elevation of MLKL, RIPK1, and RIPK3 in PTECs treated by TGF-ß1and in mice after UUO, and CA-pretreated MSCs were superior to MSCs in alleviating necroptosis. MSCs significantly reduced TNF-α and TNFR1 expression induced by TGF-ß1 in PTECs and inhibited TGF-ß1, TNF-α, and TNFR1 expression induced by UUO in mice. These effects of MSCs were significantly enhanced after CA pretreatment. Therefore, our results suggest that CA pretreatment enhances the antifibrotic activity of MSCs by inhibiting TGF-ß1/TNF-α/TNFR1 signaling-induced necroptosis.

Smad3 Mediates Diabetic Dyslipidemia and Fatty Liver in db/db Mice by Targeting PPARδ.

Transforming growth factor-ß (TGF-ß)/Smad3 signaling has been shown to play important roles in fibrotic and inflammatory diseases. However, the role of Smad3 in dyslipidemia and non-alcoholic fatty liver disease (NAFLD) in type 2 diabetes remains unclear, and whether targeting Smad3 has a therapeutic effect on these metabolic abnormalities remains unexplored. These topics were investigated in this study in Smad3 knockout (KO)-db/db mice and by treating db/db mice with a Smad3-specific inhibitor SIS3. Compared to Smad3 wild-type (WT)-db/db mice, Smad3 KO-db/db mice were protected against dyslipidemia and NAFLD. Similarly, treatment of db/db mice with SIS3 at week 4 before the onset of type 2 diabetes until week 12 was capable of lowering blood glucose levels and improving diabetic dyslipidemia and NAFLD. In addition, using RNA-sequencing, the potential Smad3-target genes related to lipid metabolism was identified in the liver tissues of Smad3 KO/WT mice, and the regulatory mechanisms were investigated. Mechanistically, we uncovered that Smad3 targeted peroxisome proliferator-activated receptor delta (PPARδ) to induce dyslipidemia and NAFLD in db/db mice, which was improved by genetically deleting and pharmacologically inhibiting Smad3.

Deletion of Smad3 protects against diabetic myocardiopathy in db/db mice.

Diabetic cardiomyopathy (DCM) is a common diabetic complication characterized by diastolic relaxation abnormalities, myocardial fibrosis and chronic heart failure. Although TGF-ß/Smad3 signalling has been shown to play a critical role in chronic heart disease, the role and mechanisms of Smad3 in DCM remain unclear. We reported here the potential role of Smad3 in the development of DCM by genetically deleting the Smad3 gene from db/db mice. At the age of 32 weeks, Smad3WT-db/db mice developed moderate to severe DCM as demonstrated by a marked increase in the left ventricular (LV) mass, a significant fall in the LV ejection fraction (EF) and LV fractional shortening (FS), and progressive myocardial fibrosis and inflammation. In contrast, db/db mice lacking Smad3 (Smad3KO-db/db) were protected against the development of DCM with normal cardiac function and undetectable myocardial inflammation and fibrosis. Interestingly, db/db mice with deleting one copy of Smad3 (Smad3 ± db/db) did not show any cardioprotective effects. Mechanistically, we found that deletion of Smad3 from db/db mice largely protected cardiac Smad7 from Smurf2-mediated ubiquitin proteasome degradation, thereby inducing IBα to suppress NF-kB-driven cardiac inflammation. In addition, deletion of Smad3 also altered Smad3-dependent miRNAs by up-regulating cardiac miR-29b while suppressing miR-21 to exhibit the cardioprotective effect on Smad3KO-db/db mice. In conclusion, results from this study reveal that Smad3 is a key mediator in the pathogenesis of DCM. Targeting Smad3 may be a novel therapy for DCM.

Decursin inhibits the growth of HeLa cervical cancer cells through PI3K/Akt signaling.

Decursin, a coumarin compound isolated from Angelica gigas has been shown to possess multiple anti-tumor activities. But it's still little known about the effects associated with cervical cancer. To explore the anti-tumor role of decursin and gain insights into its underlying mechanisms, we analyzed proliferation in parallel with apoptosis and migration in HeLa cells. Our findings implied that decursin can provoke apoptosis, and inhibit cell proliferation, migration in HeLa cells. More importantly, decursin also inhibited the tumor growth in vivo. The mechanisms may be associated with the regulation of Akt activation, with implications for novel therapeutic strategies on cervical cancer.[Formula: see text].

TGF-Beta as a Master Regulator of Diabetic Nephropathy.

Diabetic nephropathy (DN) is one of the most common complications in diabetes mellitus and the leading cause of end-stage renal disease. TGF-ß is a pleiotropic cytokine and has been recognized as a key mediator of DN. However, anti-TGF-ß treatment for DN remains controversial due to the diverse role of TGF-ß1 in DN. Thus, understanding the regulatory role and mechanisms of TGF-ß in the pathogenesis of DN is the initial step towards the development of anti-TGF-ß treatment for DN. In this review, we first discuss the diverse roles and signaling mechanisms of TGF-ß in DN by focusing on the latent versus active TGF-ß1, the TGF-ß receptors, and the downstream individual Smad signaling molecules including Smad2, Smad3, Smad4, and Smad7. Then, we dissect the regulatory mechanisms of TGF-ß/Smad signaling in the development of DN by emphasizing Smad-dependent non-coding RNAs including microRNAs and long-non-coding RNAs. Finally, the potential therapeutic strategies for DN by targeting TGF-ß signaling with various therapeutic approaches are discussed.

Spectrum of PALB2 germline mutations and characteristics of PALB2-related breast cancer: Screening of 16,501 unselected patients with breast cancer and 5890 controls by next-generation sequencing.

BACKGROUND: Partner and localizer BRCA2 (PALB2) is a breast cancer predisposition gene, but the clinical relevance of PALB2 germline mutations in Chinese patients with breast cancer remains unknown. This study attempted to investigate the full prevalence and spectrum of PALB2 germline mutations in China and the associations between PALB2 germline mutations and breast cancer risk. METHODS: A total of 21,216 unselected patients with breast cancer were enrolled from 10 provinces in China, and 5890 Chinese women without cancer were enrolled as healthy controls. PALB2 screening was based on next-generation sequencing. RESULTS: A total of 16,501 BRCA1/2-negative patients with breast cancer were analyzed. Deleterious PALB2 mutation carriers accounted for 0.97% (n = 160) in the breast cancer cohort and for 0.19% (n = 11) in the healthy control cohort. Forty-one novel PALB2 germline mutations were identified. A high frequency of PALB2 c.751C>T was detected, and it accounted for 10.63% of the PALB2 germline mutations detected (17 of 160). PALB2 mutations were significantly associated with increased breast cancer risk (odds ratio [OR], 5.23; 95% confidence interval [CI], 2.84-9.65; P < .0001), especially among women 30 years old or younger (OR, 10.09; 95% CI, 3.95-25.79; P < .0001). Clinical characteristics, including a family history, bigger tumor size, triple-negative breast cancer, positive lymph nodes, and bilateral breast cancer, were closely related to PALB2 mutations. CONCLUSIONS: This study revealed a comprehensive spectrum of PALB2 germline mutations and characteristics of PALB2-related breast cancer in China. PALB2 germline mutations confer a moderately increased risk for breast cancer but profoundly increase breast cancer risk for those 30 years old or younger in the Chinese population.

Profiling of the germline mutation BRCA1: p.Ile1845fs in a large cohort of Han Chinese breast cancer.

BACKGROUND: Breast cancer is a one of the malignant carcinomas partially caused by genetic risk factors. Germline BRCA1 gene mutations are reportedly associated with breast cancers. Identification of BRCA1 mutations greatly improves the preventive strategies and management of breast cancer. The aim of our study was to investigate the frequency of the deleterious BRCA1: p.Ile1845fs variant in breast carcinomas, as well as the correlation between p.Ile1845fs variant with clinicopathological parameters and clinical outcomes. RESULTS: A total of 23,481 clinically high-risk patients with breast cancer and 6489 healthy controls were recruited for p.Ile1845fs variant sequencing (either sanger or next generation sequencing). We identified 94 breast cancer patients (0.40%, 94/23481) as well as 11 healthy controls (0.17%, 11/6489) carried p.Ile1845fs variant. BRCA1: p.Ile1845fs variant showed a higher frequency in patients with TNBC molecular typing (20.21%, 19/94) and family history (37.23%, 35/94) compared with non-carriers (P = 3.62E-6 and 0.034, respectively). According to our data, we advanced the frequency of p.Ile1845fs variant and we confirmed that BRCA1: p.Ile1845fs variant was associated with increased risk of breast cancer (OR = 2.36, 95%CI = 1.26-4.89, P = 0.004). CONCLUSIONS: BRCA1: p.Ile1845fs variant was a frequently pathogenic mutation in breast cancer in Han Chinese women and our data may be helpful for diagnosis and therapy of breast cancer.

Alpha lipoic acid inhibits oxidative stress-induced apoptosis by modulating of Nrf2 signalling pathway after traumatic brain injury.

Alpha lipoic acid (ALA) is a powerful antioxidant which has been widely used in the treatment of different system diseases, such as cardiovascular and cerebrovascular diseases. But, there are few studies that refer to protective effects and potential mechanisms on traumatic brain injury (TBI). This study was carried out to investigate the neuroprotective effect following TBI and illuminate the underlying mechanism. Weight drop-injured model in rats was induced by weight-drop. ALA was administrated via intraperitoneal injection after TBI. Neurologic scores were examined following several tests. Neurological score was performed to measure behavioural outcomes. Nissl staining and TUNEL were performed to evaluate the neuronal apoptosis. Western blotting was engaged to analyse the protein content of the Nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream protein factors, including hemeoxygenase-1 (HO-1) and quinine oxidoreductase-1 (NQO1). ALA treatment alleviated TBI-induced neuron cell apoptosis and improved neurobehavioural function by up-regulation of Nrf2 expression and its downstream protein factors after TBI. This study presents new perspective of the mechanisms responsible for the neuronal apoptosis of ALA, with possible involvement of Nrf2 pathway.

A simple and highly purified method for isolation of glomeruli from the mouse kidney.

Highly purified mouse glomeruli are of great value for studying glomerulus-associated kidney diseases. Here, we developed a simple and rapid procedure for mouse glomerular isolation with large quantity and high purity based on the combination of size-selective sieving and differential adhesion techniques, which we termed the "differential adhesion method." In this method, mouse renal cortices were minced and digested with collagenase. Glomeruli were disassociated from tubules by successive sieving through 105-, 75-, and 40-µm cell strainers. The retained glomeruli-rich preparation on the 40-µm strainer was rinsed into a cell culture dish to allow tubules to adhere quickly to the dish while leaving most glomeruli floating (termed "differential adhesion"). The floating glomerular fraction was then subjected to another wash through the 40-µm strainer followed by an additional differential adhesion step to obtain highly purified glomeruli with yields of 8,357 ± 575 and purity of 96.1 ± 1.8% from one adult C57BL/6 mouse. The purity of the isolated glomeruli was further confirmed by high expression of the podocyte marker nephrin without detectable tubular marker cadherin-16. Importantly, we also found that although both the quantity and purity of the isolated glomeruli by this and the established Dynabeads method were comparable, glomeruli isolated by the current method showed much less inflammatory stress in terms of proinflammatory cytokine expression than the Dynabeads method. In conclusion, we established a newly mouse glomerular isolation method that is simple, rapid, cost effective, and productive. It provides an advanced methodology for research into glomerulus-related kidney diseases in the mouse.

Specific Inhibitor of Smad3 (SIS3) Attenuates Fibrosis, Apoptosis, and Inflammation in Unilateral Ureteral Obstruction Kidneys by Inhibition of Transforming Growth Factor ß (TGF-ß)/Smad3 Signaling.

BACKGROUND Fibrosis is the common pathological feature in most kinds of chronic kidney disease (CKD). TGF-ß/Smads signaling is the master pathway regulating kidney fibrosis pathogenesis, in which Smad3 acts as the integrator of various pro-fibrosis signals. In this study, we analyzed the role of SIS3, a specific inhibitor of Smad3, in mouse unilateral ureteral obstruction (UUO) kidneys. MATERIAL AND METHODS UUO mice were intraperitoneally injected with 0.2 mg/kg/day or 2 mg/kg/day of SIS3 or control saline for 7 days, followed by analysis of structure injury, fibrosis status, inflammation, apoptosis, and TGF-ß/Smads signaling activity. RESULTS Our results indicated that SIS3 treatment dosage-dependently relieved the gross structure injury and tubular necrosis in UUO kidneys. Masson staining, immunohistochemistry, and real-time PCR showed significantly decreased extracellular matrix deposition, fibronectin staining intensity, and RNA levels of collagen I and collagen III in SIS3-treated UUO kidneys. SIS3 treatment also suppressed the activation of myofibroblasts, as evidenced by decreased expression levels of a-SMA and vimentin in UUO kidneys. The TGF-ß/Smads signaling activity analysis showed that SIS3 inhibited the phosphorylation of Smad3 but not Smad2 and decreased the protein level of TGF-ß1, suggesting specific inhibition of the TGF-ß/Smad3 pathway in UUO kidneys. Furthermore, SIS3 treatment also ameliorated the increased pro-inflammatory TNF-α and COX2 in UUO kidneys and circulating IL-1ß in UUO mice, and inhibited caspase-3 activity and the number of apoptotic cells. CONCLUSIONS SIS3 ameliorated fibrosis, apoptosis, and inflammation through inhibition of TGF-b/Smad3 signaling in UUO mouse kidneys.

Decursin inhibits the growth of HepG2 hepatocellular carcinoma cells via Hippo/YAP signaling pathway.

Targeted therapy has a pivotal role for the treatment of liver cancer. The aim of this current study was to examine the effects of decursin on the growth of HepG2 cells and the underlying mechanisms. Our present study showed that treatment of HepG2 cells with decursin significantly inhibited the growth of HepG2 cells by suppressing cell proliferation, cell cycle arresting, and promoting apoptosis in a dose- and time-dependent manner. Most significantly, administration of decursin dramatically impeded in vivo tumor growth in nude mice. Mechanically, it is noteworthy that decursin treatment provoked degradation of YAP by upregulating the expression of phosphorylated LATS1 and ßTRCP. Moreover, apoptosis caused by decursin could be reversed by a selective MST1/2 inhibitor, XMU-MP-1, suggesting that decursin may function through Hippo/YAP signaling. This study has identified that decursin is a potential agent for HCC therapy, and further research should be undertaken to facilitate its therapeutic application.

Gulo Acts as a de novo Marker for Pronephric Tubules in Xenopus laevis.

BACKGROUNDS/AIMS: Vitamin C is an antioxidant and acts as a cofactor for several key enzymatic catalytic reactions in animals. Amphibians produce vitamin C in their kidneys, as opposed to mammals that produce vitamin C in their liver. Gulo serves as a crucial enzyme for vitamin C synthesis in mammals, but the characteristics and localization of its homologous genes during kidney development in Xenopus laevis, an amphibian, remains unknown. METHODS: We aligned amino acid sequences of Gulo across different species by using bioinformatics methods and detected patterns of expression for Gulo during kidney development by using RT-PCR and in situ hybridization. RESULTS: We identified a new site on the X. laevis genome, LOC495407. Sequence alignment analysis indicated this fragment is highly conserved and homologous to gulo genes in mammals. RT-PCR and in situ hybridization results reveal that X. laevis gulo is maternally expressed during the early stages of embryonic development, particularly, in the tubules of the pronephros from the middle tail-bud stage and onward in embryos. CONCLUSION: Gulo is a novel specific marker for pronephros tubules in X. laevis, and may be used as a potential marker for kidney development studies and disease diagnosis in mammals.

CagA promotes proliferation and secretion of extracellular matrix by inhibiting signaling pathway of apoptosis in rat glomerular mesangial cells.

Cytotoxin-associated antigen A (CagA), a major virulence factor of Helicobacter pylori (Hp), is associated with the pathogenesis of peptic ulcer and gastric cancer. Recent researches demonstrated that Hp exists in palatine tonsil in all studied IgA nephropathy (IgAN) patients, most of which were CagA-positive, suggesting that CagA may be a causative pathogenic factor of IgAN. However, the underlying molecular mechanisms and signaling pathway are still largely unclear. In the present study, CCK8 assay, enzyme-linked immunosorbent assay, and immunohistochemistry were performed to investigate the effect of CagA on cell proliferation and extracellular matrix secretion in rat glomerular mesangial cells. RT-PCR and western blotting were used to reveal the potential signaling pathway. Rat glomerular mesangial cells were treated with recombinant CagA protein for 72 h, in a dose- and time-dependent manner. We found that CagA promoted cell proliferation and extracellular matrix secretion by inhibiting signaling pathway of apoptosis. Taken together, these findings suggested that CagA induced cellular injury in glomerular mesangium by proliferation and secretion of extracellular matrix, and may play an important role in pathogenesis of IgAN.

Helicobacter pylori participates in the pathogenesis of IgA nephropathy.

Increasing evidences have shown that Helicobacter pylori (Hp) is a pathogen closely related to extra-gastric disorders. Our previous in vitro studies had demonstrated that Hp infection, at least via cytotoxin-associated gene A protein (CagA), might play an important role in the pathogenesis of IgA nephropathy (IgAN) by stimulating proliferation and ectopic synthesis of aberrantly glycosylated IgA1 of B cells. However, the relevant clinical evidence of IgAN resulted from Hp infection remain to be elucidated. This study aimed to investigate the risk incidence of IgAN caused by Hp infection. 22 primary IgAN, 20 non-IgA nephropathy (n-IgAN), and 30 healthy controls were included in this study. We found that the rate of IgG anti-Hp seropositivity was significantly improved in IgAN, but the current Hp infection was similar in all groups. The production and underglycosylation of IgA1 tended to increase in IgAN patients with IgG anti-Hp seropositivity. A tendency toward increased the risk of clinical prognosis was seen in IgAN with Hp infection. Hp antigen and CagA were only deposited in renal tubules, and enhanced antigen deposition in response to Hp was observed in IgAN. Our study suggested that Hp infection might have a pathogenic role in IgAN through giving rise to strongly mucosal immune response, and based on damage of renal tubular.

Apobec-1 Complementation Factor (A1CF) Inhibits Epithelial-Mesenchymal Transition and Migration of Normal Rat Kidney Proximal Tubular Epithelial Cells.

Apobec-1 complementation factor (A1CF) is a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family, which participates in site-specific posttranscriptional RNA editing of apolipoprotein B (apoB) transcript. The posttranscriptional editing of apoB mRNA by A1CF in the small intestine is required for lipid absorption. Apart from the intestine, A1CF mRNA is also reported to be highly expressed in the kidneys. However, it is remained unknown about the functions of A1CF in the kidneys. The aim of this paper is to explore the potential functions of A1CF in the kidneys. Our results demonstrated that in C57BL/6 mice A1CF was weakly expressed in embryonic kidneys from E15.5dpc while strongly expressed in mature kidneys after birth, and it mainly existed in the tubules of inner cortex. More importantly, we identified A1CF negatively regulated the process of epithelial-mesenchymal transition (EMT) in kidney tubular epithelial cells. Our results found ectopic expression of A1CF up-regulated the epithelial markers E-cadherin, and down-regulated the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) in NRK52e cells. In addition, knockdown of A1CF enhanced EMT contrary to the overexpression effect. Notably, the two A1CF variants led to the similar trend in the EMT process. Taken together, these data suggest that A1CF may be an antagonistic factor to the EMT process of kidney tubular epithelial cells.

EIYMNVPV Motif is Essential for A1CF Nucleus Localization and A1CF (-8aa) Promotes Proliferation of MDA-MB-231 Cells via Up-Regulation of IL-6.

Apobec-1 complementation factor (A1CF) is a heterogeneous nuclear ribonuceloprotein (hnRNP) and mediates apolipoprotein-B mRNA editing. A1CF can promote the regeneration of the liver by post-transcriptionally stabilizing Interleukin-6 (IL-6) mRNA. It also contains two transcriptional variants-A1CF64 and A1CF65, distinguished by the appearance of a 24-nucleotide motif which contributes to the corresponding eight-amino acid motif of EIYMNVPV. For the first time, we demonstrated that the EIYMNVPV motif was essential for A1CF nucleus localization, A1CF deficient of the EIYMNVPV motif, A1CF (-8aa) showed cytoplasm distribution. More importantly, we found that A1CF (-8aa), but not its full-length counterpart, can promote proliferation of MDA-MB-231 cells accompanied with increased level of IL-6 mRNA. Furthermore, silencing of IL-6 attenuated A1CF (-8aa)-induced proliferation in MDA-MB-231 cells. In conclusion, notably, these findings suggest that A1CF (-8aa) promoted proliferation of MDA-MB-231 cells in vitro viewing IL-6 as a target. Thus, the EIYMNVPV motif could be developed as a potential target for basal-like breast cancer therapy.

Applying a new method for direct collection, volume quantification and determination of N2 emission from water.

The emission of N2 is important to remove excess N from lakes, ponds, and wetlands. To investigate the gas emission from water, Gao et al. (2013) developed a new method using a bubble trap device to collect gas samples from waters. However, the determination accuracy of sampling volume and gas component concentration was still debatable. In this study, the method was optimized for in situ sampling, accurate volume measurement and direct injection to a gas chromatograph for the analysis of N2 and other gases. By the optimized new method, the recovery rate for N2 was 100.28% on average; the mean coefficient of determination (R(2)) was 0.9997; the limit of detection was 0.02%. We further assessed the effects of the new method, bottle full of water, vs. vacuum bag and vacuum vial methods, on variations of N2 concentration as influenced by sample storage times of 1, 2, 3, 5, and 7 days at constant temperature of 15°C, using indices of averaged relative peak area (%) in comparison with the averaged relative peak area of each method at 0 day. The indices of the bottle full of water method were the lowest (99.5%-108.5%) compared to the indices of vacuum bag and vacuum vial methods (119%-217%). Meanwhile, the gas chromatograph determination of other gas components (O2, CH4, and N2O) was also accurate. The new method was an alternative way to investigate N2 released from various kinds of aquatic ecosystems.

The association of apolipoprotein E gene polymorphisms with cerebral palsy in Chinese infants.

Apolipoprotein E (APOE, protein; ApoE, gene) is a lipid transport protein abundantly present in brain cells. Previous studies have suggested that there is an association between genetic variants of ApoE and susceptibility to cerebral palsy (CP). The purpose of this study was to explore whether the ApoE gene is involved in the etiology of CP in the Chinese population. In this study, 350 CP patients and 242 healthy control children were recruited. Genomic DNA was prepared from venous blood and all five single nucleotide polymorphisms (SNPs) in ApoE (rs769446, rs405509, rs121918399, rs429358, and rs190853081) were detected by the MassARRAY platform-based genotyping approach. The SHEsis program was used to analyze the genotyping data, and we systemically analyzed the association of the ApoE SNPs with different subtypes of CP. No significant association was detected between the e4 identified by the C allele of rs429358 and CP, but there were significant differences in allelic frequencies between the CP patients and controls at rs769446 (P = 0.005, P = 0.025 after Bonferroni correction), as well as between the CP patients with preterm birth (<34 gestational weeks) and controls at rs769446 (P = 0.001, P = 0.005 after Bonferroni correction). A haplotype consisting of the five SNPs rs769446(C), rs405509(C), rs121918399(C), rs429358(T), and rs190853081(G) was associated with a decreased risk of CP (P = 0.002 after Bonferroni correction). However, we found no significant association between any of the other three SNPs and CP based on different subgroup analyses. This study provides the first evidence that ApoE gene polymorphisms are a potential risk factor for CP in the Chinese population.

The association between sex-related interleukin-6 gene polymorphisms and the risk for cerebral palsy.

BACKGROUND: The relationship between genetic factors and the development of cerebral palsy (CP) has recently attracted much attention. Polymorphisms in the genes encoding proinflammatory cytokines have been shown to be associated with susceptibility to perinatal brain injury and development of CP. Interleukin-6 (IL-6) is a proinflammatory cytokine that plays a pivotal role in neonatal brain injury, but conflicting results have been reported regarding the association between IL-6 single nucleotide polymorphisms (SNPs) and CP. The purpose of this study was to analyze IL-6 gene polymorphisms and protein expression and to explore the role of IL-6 in the Chinese CP population. METHODS: A total of 753 healthy controls and 713 CP patients were studied to detect the presence of five SNPs (rs1800796, rs2069837, rs2066992, rs2069840, and rs10242595) in the IL-6 locus. Of these, 77 healthy controls and 87 CP patients were selected for measurement of plasma IL-6 by Luminex assay. The SHEsis program was used to analyze the genotyping data. For all comparisons; multiple testing on each individual SNP was corrected by the SNPSpD program. RESULTS: There were no differences in allele or genotype frequencies between the overall CP patients and controls among the five genetic polymorphisms. However, subgroup analysis found significant sex-related differences in allele and genotype frequencies. Differences were found between spastic CP and controls in males for rs2069837; between CP with periventricular leukomalacia and controls in males for rs1800796 and rs2066992; and between term CP and controls in males for rs2069837. Plasma IL-6 levels were higher in CP patients than in the controls, and this difference was more robust in full-term male spastic CP patients. Furthermore, the genotype has an effect on IL-6 synthesis. CONCLUSIONS: The influence of IL-6 gene polymorphisms on IL-6 synthesis and the susceptibility to CP is related to sex and gestational age.