Elevated 2-oxoglutarate antagonizes DNA damage responses in cholangiocarcinoma chemotherapy through regulating aspartate beta-hydroxylase.

Cholangiocarcinoma (CCA) is resistant to systemic chemotherapies that kill malignant cells mainly through DNA damage responses (DDRs). Recent studies suggest that the involvement of 2-oxoglutarate (2-OG) dependent dioxygenases in DDRs may be associated with chemoresistance in malignancy, but how 2-OG impacts DDRs in CCA chemotherapy remains elusive. We examined serum 2-OG levels in CCA patients before receiving chemotherapy. CCA patients are classified as progressive disease (PD), partial response (PR), and stable disease (SD) after receiving chemotherapy. CCA patients classified as PD showed significantly higher serum 2-OG levels than those defined as SD and PR. Treating CCA cells with 2-OG reduced DDRs. Overexpression of full-length aspartate beta-hydroxylase (ASPH) could mimic the effects of 2-OG on DDRs, suggesting the important role of ASPH in chemoresistance. Indeed, the knockdown of ASPH improved chemotherapy in CCA cells. Targeting ASPH with a specific small molecule inhibitor also enhanced the effects of chemotherapy. Mechanistically, ASPH modulates DDRs by affecting ATM and ATR, two of the major regulators finely controlling DDRs. More importantly, targeting ASPH improved the therapeutic potential of chemotherapy in two preclinical CCA models. Our data suggested the impacts of elevated 2-OG and ASPH on chemoresistance through antagonizing DDRs. Targeting ASPH may enhance DDRs, improving chemotherapy in CCA patients.

The multiple reference range of mean uterine artery pulsatility index for natural and in vitro fertilization singletons during 11-14 gestational weeks.

Background: Ultrasonography of the uterine artery (UtA) in the first and second trimesters of pregnancy can assess uterine-placental blood perfusion and guide early clinical prevention. Establishing normal ranges of the UtA pulsatility index (UtA-PI) at 11-14 weeks of pregnancy is helpful for the early identification of high-risk pregnant women and improving the prognosis. This study aimed to establish a reference range of UtA-PI based on crown-rump length (CRL) for spontaneous and in vitro fertilization (IVF) singleton pregnancy during 11-14 weeks, respectively. Methods: A prospective study was performed at Peking Union Medical College Hospital. Healthy, low-risk women with a singleton pregnancy at 11-14 gestational weeks were consecutively recruited for this study from December 2017 to December 2020. All participants underwent routine prenatal ultrasound examination. The CRL of the fetus and the UtA-PI were measured in both uterine arteries, and average values were calculated. The LMS method was used to fit the percentile (P)5, P10, P25, P50, P75, P90, and P95 curves of the UtA-PI value of spontaneous and IVF singleton pregnancy with CRL changes, respectively. Results: A total of 1,962 pregnant women with normal fetuses were included in this study, including 1,792 pregnancies conceived naturally and 170 IVF fetuses. The UtA-PI reference range in the spontaneous pregnancy group was consistently higher than that in the IVF group during 11-14 weeks, and showed a statistically significant difference in UtA-PI for spontaneous and IVF pregnancies (P<0.001). According to the LMS method, each percentile curve of UtA-PI decreased with the increase of CRL in both the natural pregnancy group and the IVF group. The P95 range of UtA-PI for pregnant women with naturally conceived and IVF pregnancy was 2.74 to 2.11 and 2.50 to 1.94, respectively. The overall change of UtA-PI differentials of the two groups showed a downward trend and decreased slightly with the increase of CRL. Conclusions: This study provided a single-center, large sample of data and constructed a CRL-based reference value of UtA-PI for spontaneous and IVF singleton pregnancy, which provides a reliable basis for early UtA evaluation and early clinical decision-making during 11-14 gestational weeks.

Curcumin mitigates deoxynivalenol-induced intestinal epithelial barrier disruption by regulating Nrf2/p53 and NF-κB/MLCK signaling in mice.

Deoxynivalenol (DON) induces intestinal epithelial barrier disruption, posing a threat to the body. Curcumin (Cur) possesses pharmacological bioactivities such as antioxidant and anti-inflammatory effects that help maintain intestinal health. Here, the protective effects of Cur against DON-induced intestinal epithelial barrier disruption were explored. Cur (75 or 150 mg/kg body weight [B.W.]) alleviated DON-induced (2.4 mg/kg B.W.) inhibition of growth performance and morphological damage to intestinal epithelium in mice. Cur also significantly attenuated DON-induced intestinal epithelial barrier disruption and structural damage to the tight junctions (TJs), as assessed by ultrastructure observation, serum FITC-dextran concentrations and diamine oxidase activity. Cur mitigated the DON-induced increase in reactive oxygen species, malondialdehyde and 8-hydroxy-2'-deoxyguanosine levels; p53, cytoplasmic cytochrome c, Bax, and Bcl-2 expression; and TUNEL-positive cell rate and caspase-3 activity. It decreased the total antioxidant capacity and expression of nuclear Nrf2 and its downstream target genes. Lastly, Cur attenuated the DON-induced increase in MLCK, p-MLC, nuclear NF-κB p65 expression, and the NF-κB downstream target genes; decrease in the expression of TJs proteins (claudin-1, occludin, and zonula occludens-1 [ZO-1]); and abnormal ZO-1 distribution. Overall, Cur mitigated the DON-induced disruption of the intestinal epithelial barrier by regulating the Nrf2/p53-mediated apoptotic pathway and NF-κB/MLCK-mediated TJs pathway in mice.

PINK1/Parkin-mediated mitophagy is activated to protect against testicular damage caused by aluminum.

Oxidative stress is the main mechanism of aluminum (Al) reproductive toxicity and can also lead to mitochondrial damage. Damaged mitochondria can trigger apoptosis, leading to testicular damage. PINK1 (phosphatase and tensin homolog (PTEN)-induced putative kinase1)/Parkin (E3 ubiquitin ligase PARK2)-mediated mitophagy can remove damaged mitochondria to maintain cellular homeostasis. However, its role in testicular damage caused by Al is unclear. In this study, first, 24 male wild type (WT) C57BL/6 N mice were divided into 4 groups and orally administered with 0, 44.825, 89.65, and 179.3 mg/kg body weight AlCl3 for 90 days, respectively. We demonstrated that apoptosis and PINK1/Parkin-mediated mitophagy were activated in a dose-effect relationship in the damaged testis caused by AlCl3, and were most significant at 179.3 mg/kg body weight AlCl3. Then, 40 male WT C57BL/6 N mice and 40 male Parkin knockout (Parkin-/-) C57BL/6 N mice were divided into 4 groups and orally administered with 0, 179.3, 0 or 179.3 mg/kg body weight AlCl3 for 90 days, respectively. Parkin-/- inhibited mitophagy, exacerbated the growth inhibition, testicular damage, mitochondrial damage, oxidative stress, and apoptosis under AlCl3 exposure. In general, the results reveal that AlCl3 exposure can activate PINK1/Parkin-mediated mitophagy, and the PINK1/Parkin-mediated mitophagy plays a protective role in the testicular damage caused by AlCl3.

Management of hemophagocytic lymphohistiocytosis in pregnancy: Case series study and literature review.

AIM: The diagnosis and treatment of hemophagocytic lymphohistiocytosis (HLH) in pregnancy is challenging due to its rarity. We aim to analyze and summarize the clinical characteristics of HLH in pregnancy, and to discuss effective diagnostic and treatment options. METHODS: Thirteen patients with HLH during pregnancy who were diagnosed and treated at the Peking Union Medical College Hospital of the Chinese Academy of Medical Sciences from January 2000 to December 2019 were studied retrospectively. We collected data on treatment regimens and on maternal and pregnancy outcomes. RESULTS: All patients had a singleton pregnancy, with a median age of 28 years (range, 22-33 years) and a median gestational age of 23 weeks (7-36 weeks). Twelve patients received corticosteroids, and four patients (with/without intravenous immunoglobulin) showed a curative effect. Two patients who were treated with dexamethasone and etoposide after termination of pregnancy achieved complete remission. Two patients attained remission after termination of pregnancy. Four pregnant women died, and the mortality rate was 30.8% (4/13). Fetal or neonatal death up to 1 week after delivery occurred in eight (61.5%) pregnancies. CONCLUSIONS: Early diagnosis and treatment are important for maternal survival, and corticosteroids are the first choice for most patients with HLH during pregnancy. For patients who do not respond to corticosteroids, etoposide and termination of pregnancy may be life-saving.

Association of a unicornuate uterus with adverse obstetric outcomes: A retrospective cohort study.

OBJECTIVE: To estimate the association of unicornuate uterus (UU) with adverse obstetric outcomes. METHODS: Using data from 26 737 singleton childbirths from a tertiary hospital from 1999 to 2019, we identified 44 births from women with a UU. A total of 367 births from women with a normal uterus were randomly selected as controls. The outcome measures were preterm birth (PTB), breech presentation, and cesarean delivery. The subdivisions of PTB and indications for cesarean delivery were described. RESULTS: The presence of UU was associated with an increased risk of PTB (adjusted risk ratio [aRR], 2.3; 95% confidence interval [CI], 1.1-4.9), breech presentation (aRR, 6.2; 95% CI, 2.9-13.2), and cesarean delivery (aRR, 2.1; 95% CI, 1.8-2.7). For women with a UU, most PTBs (7/9) were moderate to late PTBs, and approximately half of the PTBs (4/9) were iatrogenic due to preeclampsia (PE). Breech presentation, PE, and prior surgery for rudimentary horn resection were UU-related indications for cesarean delivery. CONCLUSIONS: Women with a UU have a higher risk of PTB, breech presentation, and cesarean delivery. Understanding of the subdivisions of PTBs and indications for cesarean delivery might help clinicians when counseling women with pregnancy complicated by a UU.

Pregnancy after the diagnosis of lymphangioleiomyomatosis (LAM).

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a rare disease that almost exclusively affects women of reproductive age. Patients are warned of the increased risks if they become pregnant. However, information on pregnancy in patients after the diagnosis of LAM is limited. METHODS: Patients were collected from the LAM registry study at Peking Union Medical College Hospital, Beijing, China. Patients with a history of pregnancy after the diagnosis of LAM were included. Medical records were reviewed, and baseline information and data during and after pregnancy were collected in May 2018. RESULTS: Thirty patients with a total of 34 pregnancies after the diagnosis of LAM were included. Livebirth, spontaneous abortion and induced abortion occurred in 10, 6 and 18 pregnancies, respectively. Sirolimus treatment was common (17/34). A total of 6/10, 5/6, and 6/18 patients with livebirths, spontaneous abortions, and induced abortions respectively, had a history of sirolimus treatment. Ten pregnancies (29.4%) had LAM-associated complications during pregnancy, including the exacerbation of dyspnea in 7 patients, pneumothorax in 3 patients (2 resulting in induced abortion and 1 successful parturition), and spontaneous bleeding of renal angiomyolipomas in 2 patients (both having successful parturition). No chylothorax was found during pregnancy. There were six pregnancies in six patients (17.6%) who had a history of livebirth after sirolimus treatment for LAM (all having successful parturition and healthy infants); two of these patients reported exacerbated dyspnea after parturition compared with before pregnancy. CONCLUSIONS: Patients with LAM, especially those taking sirolimus before pregnancy, were at a higher risk of spontaneous abortion. Complications such as pneumothorax, bleeding of renal angiomyolipoma, and exacerbated dyspnea during pregnancy were common. In patients without spontaneous abortion, sirolimus discontinuation before or during pregnancy did not lead to increased adverse neonatal outcomes.

Evaluation of maternal and fetal outcomes in pregnancy complicated with pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is an uncommon type of pulmonary hypertension (PH) disease characterized by progressive remodeling of distal pulmonary arteries. It could inevitably lead to pulmonary vascular resistance and even right ventricular failure. Biologists have explored the basic pathobiology of PAH, but its functional mechanism and effect in pregnant people remain unknown. This study was designed to investigate the maternal and fetal outcomes of pregnancy-related PAH. METHODS: Clinical data of 59 pregnant women with PAH who were admitted to Peking Union Medical College Hospital from Jan. 2000 to Dec. 2018 were retrospectively reviewed and analyzed. Multiple parameters, including age, gestational week, the New York Heart Association (NYHA) cardiac functional classification, ultrasonic cardiogram (UCG), blood test, pregnancy complications, pulmonary arterial systolic pressure, maternal and fetal outcomes, were comprehensively investigated and analyzed. RESULTS: According to the pulmonary arterial systolic pressure, all 59 pregnant women were divided into mild PAH (30-49 mmHg, n=18), moderate PAH (50-79 mmHg, n=17) and severe PAH (>79 mmHg, n=24). Five patients died, and the mortality rate was 8.5%. Compared with the mild and moderate groups, the mean gestational week, age of the pregnancy, and NYHA cardiac functional classification grade in the severe PAH group were dramatically different (all P<0.05). The incidence of pregnancy-related complications in the severe PAH group was significantly higher than those in the mild and moderate PAH groups (both P<0.05). CONCLUSIONS: The blood parameters, PAH, and NYHA cardiac functional classification grade were significantly changed before and after surgery. We found that the severity of PAH was a major factor of maternal and fetal outcomes. Strengthening the nursing care for pregnant women with PAH is of great clinical significance.

Maternal Serum Angiogenic Factor sFlt-1 to PlGF Ratio in Preeclampsia: A Useful Marker for Differential Diagnosis and Prognosis Evaluation in Chinese Women.

The ratio of soluble fms-like tyrosine kinase-1 to placental growth factor (sFlt-1/PlGF) is elevated and proved to be useful in preeclampsia (PE) diagnosis. Its value in differential diagnosis with other pregnancy complications and prediction of pregnancy duration has yet to be clarified in Chinese population. We retrospectively analyzed 118 singleton pregnancies with suspected or diagnosed PE at the Peking Union Medical College Hospital (PUMCH) in China. Among these, 62 pregnancies were diagnosed as PE (48 early onsets and 14 late onsets, with 39 and 5 severe PE, respectively), 12 gestational hypertension (GH), 15 chronic hypertension (chrHTN), 16 autoimmune diseases, and 13 pregnancies with uncomplicated proteinuria. And 76 normal pregnancies were included as control. The results showed (1) the sFlt-1/PlGF ratio in early onset PE subgroup was significantly higher than that in GH, chrHTN, and control groups; the sFlt-1/PlGF ratio in late onset PE subgroup was significantly higher than that in chrHTN and control groups, but similar as GH group; the sFlt-1/PlGF ratio was similar among GH, chrHTN, and control groups. (2) The sFlt-1/PlGF ratio was significantly increased in the PE group compared with autoimmune disease and uncomplicated proteinuria pregnancies. (3) By ROC curve analysis, the cutoff value of the sFlt-1/PlGF ratio was less than 21.5 to rule out PE and higher than 97.2 to confirm the diagnosis of PE. (4) The sFlt-1/PlGF ratio was higher in PE pregnancies delivering within 7 days than those more than 7 days, either in early onset PE or severe PE. In conclusion, we show that maternal sFlt-1/PlGF ratio is an efficient biomarker in the diagnosis and differential diagnosis of PE. This ratio can be used to predict the timing of delivery for PE pregnancies.

Genotypic and phenotypic characterization of Chinese patients with osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a rare hereditary skeletal dysplasia, characterized by recurrent fractures and bone deformity. This study presents a clinical characterization and mutation analysis of 668 patients, aiming to establish the mutation spectrum and to elucidate genotype-phenotype correlations in Chinese OI patients. We identified 274 sequence variants (230 in type I collagen encoding genes and 44 in noncollagen genes), including 102 novel variants, in 340 probands with a detection rate of 90%. Compared with 47 loss-of-function variants detected in COL1A1, neither nonsense nor frameshift variants were found in COL1A2 (p < 0.0001). The major cause of autosomal recessive OI was biallelic variants in WNT1 (56%, 20/36). It is noteworthy that three genomic rearrangements, including one gross deletion and one gross duplication in COL1A1 as well as one gross deletion in FKBP10, were detected in this study. Of ten individuals with glycine substitutions that lie towards the N-terminal end of the triple-helical region of the α1(I) chain, none exhibited hearing loss, suggesting a potential genotype-phenotype correlation. The findings in this study expanded the mutation spectrum and identified novel correlations between genotype and phenotype in Chinese OI patients.

Aspartate beta-hydroxylase promotes cholangiocarcinoma progression by modulating RB1 phosphorylation.

Cholangiocarcinoma (CCA) is a highly lethal and aggressive disease. Recently, IDH1/2 mutations have been identified in approximately 20% of CCAs which suggests an involvement of 2-oxoglutarate (2-OG) -dependent dioxygenases in oncogenesis. We investigated if the 2-OG dependent dioxygenase, aspartate beta-hydroxylase (ASPH) was important in tumor development and growth. Immunoassays were used to clarify how ASPH modulates CCA progression by promoting phosphorylation of the retinoblastoma protein (RB1). A xenograft model was employed to determine the role of ASPH on CCA growth. Knockdown of ASPH expression inhibited CCA development and growth by reducing RB1 phosphorylation. Expression of ASPH promoted direct protein interaction between RB1, cyclin-dependent kinases, and cyclins. Treatment with 2-OG-dependent dioxygenase and ASPH inhibitors suppressed the interaction between RB1 and CDK4 as well as RB1 phosphorylation. Knockdown of ASPH expression inhibited CCA progression and RB1 phosphorylation in vivo and they were found to be highly expressed in human CCAs. Knockdown of ASPH expression altered CCA development by modulating RB1 phosphorylation, as one of the major factors regulating the growth of these tumors.

Acetylation within the N- and C-Terminal Domains of Src Regulates Distinct Roles of STAT3-Mediated Tumorigenesis.

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation.Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825-38. ©2018 AACR.

Large area and broadband ultra-black absorber using microstructured aluminum doped silicon films.

A large area and broadband ultra-black absorber based on microstructured aluminum (Al) doped silicon (Si) films prepared by a low-cost but very effective approach is presented. The average absorption of the absorber is greater than 99% within the wide range from 350 nm to 2000 nm, and its size reaches to 6 inches. We investigate the fabrication mechanism of the absorber and find that the Al atom doped in silicon improves the formation of the nanocone-like microstructures on the film surface, resulting in a significant decrease in the reflection of incident light. The absorption mechanism is further discussed by experiments and simulated calculations in detail. The results show that the doped Al atoms and Mie resonance formed in the microstructures contribute the broadband super-high absorption.

Aspartate-ß-hydroxylase (ASPH): A potential therapeutic target in human malignant gliomas.

BACKGROUND: Despite therapeutic advances, survival with glioblastoma multiforme (GBM) remains below 15 months from diagnosis due to GBM's highly infiltrative nature which precludes complete surgical resection. Patient outcomes could potentially be improved by targeting genes and pathways that drive neoplastic cell motility and invasiveness, including hypoxia-inducible factor-1 (HIF-1α), NOTCH, and aspartate-ß-hydroxylase (ASPH). METHODS: Human astrocytoma biopsy specimens (n = 37), WHO Grades II-IV, were analyzed for levels and distributions of ASPH and HIF-1α immunoreactivity by immunohistochemical staining, and ASPH, Notch, JAG, HES1, HEY1 and HIF1α mRNA expression by quantigene multiplex analysis. The effects of small molecule inhibitors on ASPH's catalytic activity, cell viability and directional motility were examined in vitro in established GBM cell lines and primary tumor cells from an invasive mouse model of GBM. RESULTS: The highest grade astrocytoma, i.e. GBM was associated with the highest levels of ASPH and HIF1α, and both proteins were more abundantly distributed in hypoxic compared with normoxic regions of tumor. Furthermore, mining of the TCGA database revealed higher levels of ASPH expression in the mesenchymal subtype of GBM, which is associated with more aggressive and invasive behavior. In contrast, lower grade astrocytomas had low expression levels of ASPH and HIF1α. In vitro experiments demonstrated that small molecule inhibitors targeting ASPH's catalytic activity significantly reduced GBM viability and directional motility. Similar effects occurred in GBM cells that were transduced with a lentiviral sh-ASPH construct. CONCLUSION: This study demonstrates that increased ASPH expression could serve as a prognostic biomarker of gliomas and may assist in assigning tumor grade when biopsy specimens are scant. In addition, the findings suggest that GBM treatment strategies could be made more effective by including small molecule inhibitors of ASPH.

Novel aluminum plasmonic absorber enhanced by extraordinary optical transmission.

We report a theoretical and experimental study on a novel type of aluminum super absorber which exhibits a near perfect absorption based on the surface plasmon resonance in the visible and near-infrared spectrum. The absorber consists of Ag/SiO2/Al triple layers in which the top Al layer is patterned by a periodic nano hole array. The absorption spectrum can be easily controlled by adjusting the structure parameters including the radius of the nano hole and the maximal absorption can reach 99.0% in theory. We completely analyze the SPP and LSP modes supported by the metal-dielectric-metal structure and their contribution to the ultrahigh absorption. On this basis, we find a novel method to enhance the absorption via the simultaneous excitation of SPP at different interfaces theoretically and experimentally. Moreover, for the first time we clarify the EOT caused by the nano hole array can enhance the absorption by experiment, which is not reported in previous works. This kind of absorber can be fabricated by low-cost colloidal sphere lithography and the use of stable Al overcomes the disadvantages brought by the noble metal, which make it a more appropriate candidate for photovoltaics, spectroscopy, photodetectors, sensing, and surface enhanced Raman scattering.

Serum levels of leptin, adiponectin and resistin in relation to clinical characteristics in normal pregnancy and preeclampsia.

BACKGROUND: Alterations in serum adipokines in preeclampsia remain vague. We investigated the roles of leptin, adiponectin and resistin and their relationships with clinical characteristics in normotensive and preeclamptic patients. METHODS: A case-control study was carried out in a cohort of 74 preeclampsia(PE) and 79 healthy pregnant women. Serum levels of leptin, adiponectin and resistin were measured by enzyme-linked immunosorbent assay. RESULTS: The mean body mass index(BMI), the serum leptin and resistin levels were significantly higher in the PE group than in the control group (p<0.001). The resistin/creatinine ratio was also higher in the PE group than in the control group (p=0.018). No significant difference was observed in the serum adiponectin level between both groups. Serum leptin levels were positively correlated with BMI (r=0.301, p<0.001) and negatively correlated with newborn birth weight (r=-0.435, p<0.001). Serum resistin levels were also negatively correlated with birth weight (r=-0.229, p=0.005) but were unrelated to BMI. Logistic regression showed that BMI≥28 and serum leptin levels were independent factors of PE. Leptin was a potential mediator between BMI and PE (p<0.001), and the mediating effect accounted for 22.54% of the total effect. CONCLUSIONS: Leptin, resistin, and obesity play important roles in the onset of PE. Leptin and resistin may have some impacts on the fetal growth and development.

Potential Role of Phosphorylation as a Regulator of Aspartyl-(asparaginyl)-ß-hydroxylase: Relevance to Infiltrative Spread of Human Hepatocellular Carcinoma.

Abundant expression of aspartyl-(asparaginyl)-ß-hydroxylase (AAH) correlates with infiltrative growth of hepatocellular carcinoma (HCC). Herein, we examine the role of phosphorylation in relation to AAH's protein expression, hydroxylase activity, promotion of cell motility, and activation of Notch signaling in human Huh7 hepatoma cells. Predicted glycogen synthase kinase-3ß (GSK-3ß), protein kinase A (PKA), protein kinase C (PKC), and casein kinase 2 (CK2) phosphorylation sites encoded by human AAH cDNA were ablated by S/T→A site-directed mutagenesis using N-Myc-tagged constructs in which gene expression was controlled by a cytomegalovirus promoter. Functional consequences were assessed in transiently transfected Huh7 cells. Cells transfected with wildtype AAH had significantly increased AAH expression, catalytic activity, HES-1 expression, and directional motility relative to controls. Single phosphorylation site mutations in the C-terminus largely abrogated these effects and further inhibited catalytic activity relative to that in cells transfected with empty vector, whereas the effects of single point mutations within the N-terminus were more varied. In contrast, AAH cDNAs carrying multiple phosphorylation site mutations exhibited wildtype levels of AAH catalytic activity suggesting that the effects of AAH phosphorylation are complex and non-uniform. AAH expression and function can be modulated by direct phosphorylation of the protein. These findings suggest additional strategies for inhibiting infiltrative growth of HCC.

Regulation of Aspartyl-(Asparaginyl)-ß-Hydroxylase Protein Expression and Function by Phosphorylation in Hepatocellular Carcinoma Cells.

BACKGROUND: Asparaginyl-ß-hydroxylase (AAH) promotes cell adhesion, migration, and invasion via Notch activation. AAH's expression is up-regulated by insulin/IGF signaling through PI3K-Akt, but its protein is independently regulated by GSK-3ß. The multiple predicted GSK-3ß phosphorylation sites suggest post-translational mechanisms may regulate AAH protein expression. METHODS: Human Huh7 hepatoma cells were transfected with recombinant plasmids that expressed full-length N-terminal Myc-tagged (N-Myc-AAH) or C-terminal HA-tagged (C-HA-AAH) cDNA. Effects of IGF-1 on AAH protein were examined using cellular ELISAs, immunofluorescence, and Western blotting. Effects of kinase inhibitors relevant to AAH's predicted phosphorylation sites were studied. RESULTS: IGF-1 stimulation increased AAH protein expression and shifted AAH's localization from the perinuclear zone to the cell periphery, including podocytes. Subsequently, Notch-1 intracellular domain was translocated to the nucleus, which is critical for Notch- modulated gene expression. Besides GSK-3ß, inhibition of PKC, PKA, and CK2, which could potentially phosphorylate AAH, increased IGF-1 stimulated AAH protein. Finally, insulin and LiCl independently and additively increased long-term AAH protein expression. CONCLUSION: Insulin/IGF-1 stimulation of AAH and Notch are enhanced by inhibiting kinases that could phosphorylate AAH protein. Targeted manipulation of AAH's phosphorylation state may have therapeutic value for reducing AAH-Notch activation and attendant infiltrative growth of hepatocellular carcinomas.

Association study of the miRNA-binding site polymorphisms of CDKN2A/B genes with gestational diabetes mellitus susceptibility.

AIMS: Gestational diabetes mellitus (GDM) is a complex disease induced by a combination of genetic factors and environmental exposures. Growing evidence suggests that common single nucleotide polymorphisms within miRNA-binding sites (miR-binding SNPs) contribute to the development of various diseases. However, the roles of miR-binding SNPs in GDM have not been fully elucidated. The CDKN2A/B genes have been identified as two of the strongest genetic determinants for diabetes risk. The aim of the study was to first investigate the associations between miR-binding SNPs of CDKN2A/B, GDM susceptibility, and quantitative metabolism traits. METHODS: Three miR-binding SNPs of CDKN2A/B gene (rs1063192, rs3217992, and rs3088440) were selected and genotyped using TaqMan allelic discrimination assays in 839 cases of GDM and 900 controls. RESULTS: The CC genotype of CDKN2B rs1063192, which is located in the hsa-miR-323b-5p binding site, was significantly associated with GDM [OR 1.418 (1.143, 1.908); p = 0.003]. The C allele of rs1063192 occurred with significantly higher frequency in GDM [OR 1.22 (1.03, 1.44); p = 0.021]. The rs1063192 genotype CC exhibited increased glucose levels at 1 h and 3 h, as well as higher insulin levels at 3 h during an OGTT compared with the control TT genotype (p < 0.05). We also found that the rs1063192 CC genotype was associated with lower total cholesterol and LDL cholesterol levels (p < 0.05). CONCLUSIONS: The CC genotype of CDKN2B rs1063192 in the hsa-miR-323b-5p binding site increased the risk of GDM in pregnant Chinese Han women. Importantly, our study provides evidence that miR-binding SNPs are a novel source of GDM susceptibility loci.

[Value of second trimester maternal serum sFlt-1, PlGF and their ratio in the prediction of preeclampsia].

OBJECTIVE: To study the value of second trimester maternal serum soluble Fms-like tyrosine kinase 1 (sFlt-1), placenta grouth factor (PlGF) and their ratio in the prediction of preeclampsia. METHODS: In this nested case-control study, we collected second trimester maternal serum samples at 15-20 weeks and 24-28 weeks of gestation from those who developed gestational hypertensive disorders. Maternal serum sFlt-1 and PlGF were measured by electrochemiluminescence immunoassay on an automated platform. The value of sFlt-1, PlGF and their ratio were compared between gestational hypertensive group and the control group. RESULTS: Totally 41 patients with preeclampsia, 44 patients with gestational hypertension and 88 women with normal pregnancy outcomes were included in this study. There was no difference of age, gravidity, parity and preconception body mass index (BMI) between these three groups (P > 0.05) .Gestational week at delivery and neonatal birth weight were different between preeclampsia group and the control group (P < 0.01). The mean value of sFlt-1, PlGF and sFlt-1/PlGF ratio was (1 658 ± 488) µg/L, (141 ± 80) µg/L and 17 ± 9 in preeclampsia group, (1 945 ± 575) µg/L, (143 ± 52) µg/L and 15 ± 6 in gestational hypertension group, and (2 084 ± 741) µg/L, (65 ± 58) µg/L and 16 ± 9 in the control group at 15-20 weeks of gestation. There was no difference of sFlt-1, PlGF value and their ratio among the three groups at 15-20 weeks of gestation (P > 0.05) . The median value of sFlt-1, PlGF and sFlt-1/PlGF ratio were 8 525 µg/L, 35 µg/L and 398.0 in preeclampsia group, 905 µg/L, 336 µg/L and 2.7 in gestational hypertension group, 1 028 µg/L, 477 µg/L and 2.3 in the control group at 24-28 weeks of gestation. The value of sFlt-1, PlGF and their ratio was significantly different between preeclampsia group and the control group at 24-28 weeks of gestation (P < 0.01) . PlGF value was different between gestational hypertension group and the control group (P < 0.01) . The sensitivity and specificity of serum sFlt-1, PlGF and sFlt-1/PlGF ratio at 24-28 weeks of gestation to predict preeclampsia were 93% and 99% for sFlt-1 (cut off value 2 500 µg/L), 92% and 77% for PlGF (cut off value 270 µg/L) , 89% and 99% for sFlt-1/PlGF ratio (cut off value 11) . CONCLUSION: The value of sFlt-1, PlGF and their ratio at 24-28 weeks of gestation was significantly changed before clinical onset of preeclampsia.Use these serum indicators to predict preeclampsia will hopefully provide objective evidence for the management of patients who will develop preeclampsia later.