[Clinical characteristics of epileptic seizure in neurofibromatosis type 1 in 15 cases].

Objective: To summarize the clinical characteristics of epileptic seizure associated with neurofibromatosis type 1 (NF1). Methods: From January 2017 to July 2023 at Children's Hospital Capital Institute of Pediatrics, medical records of patients with both NF1 and epileptic seizure were reviewed in this case series study. The clinical characteristics, treatment and prognosis were analyzed retrospectively. Results: A total of 15 patients(12 boys and 3 girls) were collected. Café-au-lait macules were observed in all 15 patients. There were 6 patients with neurodevelopmental disorders and the main manifestations were intellectual disability or developmental delay. The age at the first epileptic seizure was 2.5 (1.2, 5.5) years. There were various seizure types, including generalized tonic-clonic seizures in 8 patients, focal motor seizures in 6 patients, epileptic spasm in 4 patients, tonic seizures in 1 patient, absence in 1 patient, generalized myoclonic seizure in 1 patient and focal to bilateral tonic-clonic seizure in 1 patient. Among 14 patients whose brain magnetic resonance imaging results were available, there were abnormal signals in corpus callosum, basal ganglia, thalamus or cerebellum in 6 patients, dilated ventricles of different degrees in 3 patients, blurred gray and white matter boundary in 2 patients, agenesis of corpus callosum in 1 patient and no obvious abnormalities in the other patients. Among 13 epilepsy patients, 8 were seizure-free with 1 or 2 antiseizure medications(ASM), 1 with drug resistant epilepsy was seizure-free after left temporal lobectomy, and the other 4 patients who have received 2 to 9 ASM had persistent seizures. One patient with complex febrile convulsion achieved seizure freedom after oral administration of diazepam on demand. One patient had only 1 unprovoked epileptic seizure and did not have another seizure without taking any ASM. Conclusions: The first epileptic seizure in NF1 patients usually occurs in infancy and early childhood, with the main seizure type of generalized tonic-clonic seizure and focal motor seizure. Some patients have intellectual disability or developmental delay. Most epilepsy patients achieve seizure freedom with ASM.

A subduction influence on ocean ridge basalts outside the Pacific subduction shield.

The plate tectonic cycle produces chemically distinct mid-ocean ridge basalts and arc volcanics, with the latter enriched in elements such as Ba, Rb, Th, Sr and Pb and depleted in Nb owing to the water-rich flux from the subducted slab. Basalts from back-arc basins, with intermediate compositions, show that such a slab flux can be transported behind the volcanic front of the arc and incorporated into mantle flow. Hence it is puzzling why melts of subduction-modified mantle have rarely been recognized in mid-ocean ridge basalts. Here we report the first mid-ocean ridge basalt samples with distinct arc signatures, akin to back-arc basin basalts, from the Arctic Gakkel Ridge. A new high precision dataset for 576 Gakkel samples suggests a pervasive subduction influence in this region. This influence can also be identified in Atlantic and Indian mid-ocean ridge basalts but is nearly absent in Pacific mid-ocean ridge basalts. Such a hemispheric-scale upper mantle heterogeneity reflects subduction modification of the asthenospheric mantle which is incorporated into mantle flow, and whose geographical distribution is controlled dominantly by a "subduction shield" that has surrounded the Pacific Ocean for 180 Myr. Simple modeling suggests that a slab flux equivalent to ~13% of the output at arcs is incorporated into the convecting upper mantle.

[The anti-proliferative and anti-inflammatory mechanisms of JAK1 inhibitor SHR0302 versus Ruxolitinib in SET2 cell line and primary cells].

Objective: To explore the effects and molecular mechanism of the selective JAK1inhibitor SHR0302 and Ruxolitinib on myeloproliterative neoplasms (MPN) cell line SET2 and primary cells in vitro. Methods: Cell proliferation was detected by CCK8 kit. Colony forming experiment was conducted to evaluate erythroid burst colony formation unit (BFU-E) of primary cells from MPN patients. Multi-factor kits were used to detect six inflammatory cytokines. Phosphorylated proteins of Jak-Stat signaling pathway were tested by Western blot. Results: At different time points after treated with SHR0302 and Ruxolitinib, the inhibition of cell proliferation was dose dependent by both drugs (P<0.01) . The inhibitory rates of 2.5 µmol/L SHR0302 and 0.1 µmol/L Ruxolitinib on SET2 cells for 72 h were comparable, i.e. (59.94±0.60) % and (64.00±0.66) %, respectively, suggesting that the inhibitory effect of SHR0302 was weaker than that of Ruxolitinib. Similarly, both SHR0302 and Ruxolitinib inhibited BFU-E in primary marrow cells from MPN patients in a dose-dependent manner. SHR0302 1.0 µmol/L produced similar degree of inhibition compared to Ruxolitinib 0.2 µmol/L. Except IL-12, the expression of other 5 cytokines (IL-6, TNF-α, IL-1ß, IL-2, IL-8) was significantly inhibited by 1.6 µmol/L SHR0302 in SET2 cells at 24 h (P<0.01) , while Ruxolitinib 1.0 µmol/L had the same effect. Several phosphorylated molecules of Jak-Stat signaling pathway were significantly inhibited by SHR0302 in SET2 cells only for 3 h. P-stat1 (Tyr701) , p-stat3 (Tyr705) were down-regulated when treated with SHR0302 1.0 µmol/L (P<0.05) , p-jak1 (tyr1022/1023) and p-stat5 (Tyr694) were inhibited at 5.0 µmol/L (P<0.05) . Ruxolitinib significantly inhibited the downstream STAT protein at 0.1 µmol/L. Again, the inhibitory effect of SHR0302 on protein expression was weaker than that of Ruxolitinib. Conclusion: SHR0302 can effectively inhibit the proliferation of MPN cell line and patients' primary cells, as well as the expression of inflammatory factors. The molecular mechanism is possibly related to the down-regulation of phosphorylated proteins of Jak-Stat signaling pathway. Overall, the anti-proliferative and anti-inflammatory effects of SHR0302 are weaker than those of Ruxolitinib.

PARP1 enhances lung adenocarcinoma metastasis by novel mechanisms independent of DNA repair.

The role of poly (ADP-ribose) polymerase 1 (PARP1) in cancer has been extensively studied in the context of DNA repair, leading to clinical trials of PARP1 inhibitors in cancers defective in homologous recombination. However, the DNA repair-independent roles of PARP1 in carcinogenesis and metastasis, particularly in lung cancer metastasis, remain largely uncharacterized. Here, we report that PARP1 promotes lung adenocarcinoma relapse to the brain and bones by regulating several steps of the metastatic process in a DNA repair-independent manner. We find that PARP1 expression is associated with overall and distant metastasis-free survival in lung adenocarcinoma patients. Consistent with this, genetic knockdown and pharmacological inhibition of PARP1 significantly attenuated the metastatic potential of lung adenocarcinoma cells. Further investigation revealed that PARP1 potentiates lung adenocarcinoma metastasis by promoting invasion, anoikis resistance, extravasation and self-renewal of lung adenocarcinoma cells and also by modifying the brain microenvironment. Finally, we identified S100A4 and CLDN7 as novel transcriptional targets and clinically relevant effectors of PARP1. Collectively, our study not only revealed previously unknown functions of PARP1 in lung adenocarcinoma metastasis but also delineated the molecular mechanisms underlying the pro-metastatic function of PARP1. Furthermore, these findings provide a foundation for the potential use of PARP1 inhibitors as a new treatment option for lung adenocarcinoma patients with elevated PARP1 expression.

Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening systemic illness of abrupt onset and unknown cause. Proteolysis of the blood-clotting protein von Willebrand factor (VWF) observed in normal plasma is decreased in TTP patients. However, the identity of the responsible protease and its role in the pathophysiology of TTP remain unknown. We performed genome-wide linkage analysis in four pedigrees of humans with congenital TTP and mapped the responsible genetic locus to chromosome 9q34. A predicted gene in the identified interval corresponds to a segment of a much larger transcript, identifying a new member of the ADAMTS family of zinc metalloproteinase genes (ADAMTS13). Analysis of patients' genomic DNA identified 12 mutations in the ADAMTS13 gene, accounting for 14 of the 15 disease alleles studied. We show that deficiency of ADAMTS13 is the molecular mechanism responsible for TTP, and suggest that physiologic proteolysis of VWF and/or other ADAMTS13 substrates is required for normal vascular homeostasis.

Drosophila LAR regulates R1-R6 and R7 target specificity in the visual system.

Different classes of photoreceptor neurons (R cells) in the Drosophila compound eye connect to specific targets in the optic lobe. Using a behavioral screen, we identified LAR, a receptor tyrosine phosphatase, as being required for R cell target specificity. In LAR mutant mosaic eyes, R1-R6 cells target to the lamina correctly, but fail to choose the correct pattern of target neurons. Although mutant R7 axons initially project to the correct layer of the medulla, they retract into inappropriate layers. Using single cell mosaics, we demonstrate that LAR controls targeting of R1-R6 and R7 in a cell-autonomous fashion. The phenotypes of LAR mutant R cells are strikingly similar to those seen in N-cadherin mutants.

Spontaneous thrombosis in mice carrying the factor V Leiden mutation.

A polymorphism in coagulation factor V, factor V Leiden (FVL), is the major known genetic risk factor for thrombosis in humans. Approximately 10% of mutation carriers experience clinically significant thrombosis in their lifetime. In a small subset of patients, thrombosis is associated with coinheritance of other prothrombotic gene mutations. However, the potential contribution of additional genetic risk factors in the majority of patients remains unknown. To gain insight into the molecular basis for the variable expressivity of FVL, mice were generated carrying the homologous mutation (R504Q [single-letter amino acid codes]) inserted into the endogenous murine Fv gene. Adult heterozygous (FvQ/+) and homozygous (FvQ/Q) mice are viable and fertile and exhibit normal survival. Compared with wild-type mice, adult FvQ/Q mice demonstrate a marked increase in spontaneous tissue fibrin deposition. No differences in fetal development or survival are observed among FvQ/Q, FvQ/+ or control littermates on the C57BL/6J genetic background. In contrast, on a mixed 129Sv-C57BL/6J genetic background, FvQ/Q mice develop disseminated intravascular thrombosis in the perinatal period, resulting in significant mortality shortly after birth. These results may explain the high degree of conservation of the R504/R506 activated protein C cleavage site within FV among mammalian species and suggest an important contribution of other genetic factors to the thrombosis associated with FVL in humans. (Blood. 2000;96:4222-4226)

Degradation of a fluoropyridinyl drug in capsule formulation: degradant identification, proposed degradation mechanism, and formulation optimization.

The purpose of this paper was to investigate the degradation chemistry of a fluoropyridinyl drug candidate in capsule formulation and to optimize the formulation based on a proposed degradation mechanism. Small developmental batches of capsules were made by tituration of drug substance and excipients using a mortar and pestle, followed by manual encapsulation. Degradants were identified by LC-MS/MS and LC-photodiode array detector (PDA) and were monitored by LC-ultraviolet detector (UVD) during stability studies. It was found that the drug could undergo a nucleophilic substitution reaction in which hydroxyl groups replace the fluorine substituents on the pyridine rings. The initial degradation rate is independent of the drug concentration but dependent on the temperature, the pH of the microenvironment, and the excipient type. On the basis of these experimental results, a nucleophilic substitution reaction mechanism for the degradation was proposed and a successful capsule formulation was developed.

Quantitation of simvastatin and its beta-hydroxy acid in human plasma by liquid-liquid cartridge extraction and liquid chromatography/tandem mass spectrometry.

A sensitive and reliable procedure for the simultaneous determination of simvastatin (SV) and its active beta-hydroxy acid metabolite (SVA) in human plasma was developed and validated. The analytes were extracted simultaneously from 0.5 ml aliquots of human plasma samples by methyl tert-butyl ether (MTBE) via Chem Elut cartridge extraction [also called liquid-solid extraction (LSE) or liquid-liquid cartridge extraction (LLCE)], separated through a Kromasil C(18) column (50 x 2 mm i.d. 5 microm) and detected by tandem mass spectrometry with a turbo ionspray interface. Stable isotope-labeled SV and SVA, (13)CD(3)-SV and (13)CD(3)-SVA, were used as internal standards. SV and SVA were detected in positive and negative ion modes, respectively, via within-run polarity switching. The use of Chem Elut cartridges not only provided a simple and efficient means of plasma sample extraction but also successfully reduced the interconversion between SV and SVA to an undetectable (for lactonization of SVA) or negligible (<0.07%, for hydrolysis of SV) level. The method showed excellent reproducibility, with intra- and inter-assay precisions <4.5% (RSD), and intra- and inter-assay accuracy between 94% and 107% of nominal values, for both analytes. The extraction recoveries were 78% and 87% on average for SV and SVA, respectively. The analyte was found to be stable in plasma through three freeze (-70 degrees C)-thaw (4 degrees C) cycles and for at least 3 h under bench-top storage condition in an ice-bath (4 degrees C), and also in the reconstitution solution at 4 degrees C for at least 24 h. The method has a lower limit of quantitation (LOQ) of 50 pg ml(-1) with a linear calibration range of 0.05-50 ng ml(-1) for both analytes, and has proved to be very reliable for the analysis of clinical samples.

Toxicity of pyroglutaminated amyloid beta-peptides 3(pE)-40 and -42 is similar to that of A beta1-40 and -42.

An N-terminal truncated isoform of the amyloid beta-peptide (A beta) that begins with a pyroglutamate (pE) residue at position 3 [A beta3(pE)-42] is the predominant isoform found in senile plaques. Based upon previous in vitro studies regarding A beta N-terminal truncated isoforms, it has been hypothesized that A beta3(pE)-x isoforms may aggregate more rapidly and become more toxic than corresponding Abeta1-x peptides. However, the toxicity and aggregation properties of A beta3(pE)-42 and A beta3(pE)-40 have not previously been examined. After initial solubilization and 1-week preaggregation of each peptide at 37 degrees C and pH 7.4, the toxicity of 5-50 microM A beta3(pE)-42 was similar to that of A beta1-42. Moreover, the toxicity of A beta3(pE)-40 paralleled that induced by A beta1-40 in both 1 day in vitro (DIV) cortical and 7 DIV hippocampal cells. Circular dichroism spectra did not reveal major differences in secondary structure between aged A beta1-42, A beta3(pE)-42, A beta3(pE)-40, and A beta1-40 or freshly solubilized forms of these peptides. Overall, the data indicate that the loss of the two N-terminal amino acids and the cyclization of glutamate at position 3 do not alter the extracellular toxicity of A beta.

The plasminogen activator inhibitor-2 gene is not required for normal murine development or survival.

Plasminogen activator inhibitor-2 (PAI-2), a member of the serpin gene family, is thought to serve as a primary regulator of plasminogen activation in the extravascular compartment. High levels of PAI-2 are found in keratinocytes, monocytes, and the human trophoblast, the latter suggesting a role in placental maintenance or embryo development. The primarily intracellular distribution of PAI-2 also may indicate a unique regulatory role in a protease-dependent cellular process such as apoptosis. To examine the potential functions of PAI-2 in vivo, we generated PAI-2-deficient mice by gene targeting in embryonic stem cells. Homozygous PAI-2-deficient mice exhibited normal development, survival, and fertility and were also indistinguishable from normal controls in response to a bacterial infectious challenge or endotoxin infusion. No differences in monocyte recruitment into the peritoneum were observed after thioglycollate injection. Epidermal wound healing was equivalent among PAI-2 -/- null and control mice. Finally, crossing PAI-2 -/- with PAI-1 -/- mice to generate animals deficient in both plasminogen activator inhibitors failed to uncover an overlap in function between these two related proteins.

Saturation mutagenesis of the plasminogen activator inhibitor-1 reactive center.

Plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of the serine proteases tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). To systematically investigate the roles of the reactive center P1 and P1' residues in PAI-1 function, saturation mutagenesis was utilized to construct a library of PAI-1 variants. Examination of 177 unique recombinant proteins indicated that a basic residue was required at P1 for significant inhibitory activity toward uPA, whereas all substitutions except proline were tolerated at P1'. P1Lys variants exhibited lower inhibition rate constants and greater sensitivity to P1' substitutions than P1Arg variants. Alterations at either P1 or P1' generally had a larger effect on the inhibition of tPA. A number of variants that were relatively specific for either uPA or tPA were identified. P1Lys-P1'Ala reacted 40-fold more rapidly with uPA than tPA, whereas P1Lys-P1'Trp showed a 6.5-fold preference for tPA. P1-P1' variants containing additional mutations near the reactive center demonstrated only minor changes in activity, suggesting that specific amino acids in this region do not contribute significantly to PAI-1 function. These findings have important implications for the role of reactive center residues in determining serine protease inhibitor (serpin) function and target specificity.

Study of root uptake and xylem translocation of cinmethylin and related compounds in detopped soybean roots using a pressure chamber technique.

A pressure chamber technique was used to study the root uptake and xylem translocation of nonradiolabeled cinmethylin and its analogs in detopped soybean (Glycine max) roots. Quantifications of compounds were achieved by gas chromatography analysis using a mass spectrometry detector under selected ion monitoring. The compounds tested, with octanol-water partition coefficients (log Kow values) ranging from 0.96 to 5.3, were all nonionizable under the experimental conditions. Root efflux curves of all compounds exhibited a steady-state kinetic profile. The time required to achieve the steady state efflux concentration in the xylem sap correlated with log Kow values in a manner very similar to the root binding profile reported previously by GG Briggs et al. ([1982] Pestic Sci 13: 495-504). After reaching the steady state efflux, the concentration ratio of each compound in the xylem sap to the final concentration in the pressure chamber was taken as the transpiration stream concentration factor (TSCF). A nonlinear relationship was observed between TSCF and log Kow values. The highest TSCF value was between 0.6 to 0.8 for compounds with log Kow between 2.5 to 3.5. The range of optimal log Kow values was slightly higher than that reported earlier by Briggs et al. ([1982] Pestic Sci 13: 495-504). After taking into account the binding of the compound to soil, the apparent optimal Kow value for best root-to-shoot translocation is lowered to around 1. The relationship of root-to-shoot and phloem translocation was also discussed to promote a better understanding at the whole plant level of the uptake and translocation of a soil-applied xenobiotic.

Molecular basis of human von Willebrand disease: analysis of platelet von Willebrand factor mRNA.

von Willebrand disease (vWD), the most common inherited bleeding disorder in humans, can result from either a quantitative or a qualitative defect in the adhesive glycoprotein, von Willebrand factor (vWF). Molecular studies of vWD have been limited by the large size of the vWF gene and difficulty in obtaining the vWF mRNA from patients. By use of an adaptation of the polymerase chain reaction, vWF mRNA was amplified and sequenced from peripheral blood platelets. A silent vWF allele was identified, resulting from a cis defect in vWF mRNA transcription or processing. In two type IIA vWD patients, two different but adjacent missense mutations were identified, the locations of which may identify an important vWF functional domain. Expression in heterologous cells of recombinant vWF containing one of these latter mutations reproduced the characteristic structural abnormality seen in type IIA vWD plasma.

Human neutrophil laminin receptors: activation-dependent receptor expression.

Activated human polymorphonuclear leukocytes (PMN) isolated from peripheral blood specifically bind 125I-laminin after stimulation with phorbol 12-myristate 13-acetate (PMA) or f-Met-Leu-Phe (FMLP) at 37 degrees C. Changes in laminin receptor expression are stimulus dose dependent at both chemotactic (10(-10) M to 10(-6) M) concentrations of FMLP, and secretory (greater than 5 ng/ml) levels of PMA. In the presence of cytochalasin B (5 micrograms/ml), 10(-7) M FMLP activation stimulates specific laminin binding, with an apparent Kd = 3.9 X 10(-9) M and 6.47 X 10(5) binding sites/cell, reaching equilibrium within 10 min at 4 degrees C. This observed activation-dependent change in laminin receptor expression is not due to interference by endogenous laminin, because no fluorescein-visualized anti-laminin antibody bound to cells without added glycoprotein, regardless of the level of activation. Levels of neutrophil lysozyme release, which show a PMA dose dependence similar to that of receptor binding activity, suggest that granule-plasma membrane fusion may be significant during increases in receptor expression. A lack of receptor stimulation by PMA from a granule-deficient patient or in granule-depleted cytoplasts from normal donors additionally supports this hypothesis. Electroblot transfer and autoradiography of subcellular fractions from unstimulated PMN reveals the presence of a 68,000 dalton laminin-binding component in the secondary/tertiary granule (beta) fraction, which may represent an intracellular laminin receptor pool.

cDNA cloning of human plasminogen activator-inhibitor from endothelial cells.

Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HUVEC) lambda gt11 cDNA library. Three overlapping clones were identified by immunologic screening of 10(6) recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-1 mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-1 gene is located on human chromosome 7.

Bile salts as atypical surfactants and solubilizers.

Recent research has suggested that self-association of bile salts does not follow the micellar pattern of self-association exhibited by typical flexible chain surfactants and detergents. A working model for the self-association of bile salts is proposed. It includes a mild degree of cooperativity in the early stages of the growth of aggregates and coexistence of a number of aggregates of different aggregation numbers. The polydispersity implies an increase in the average aggregation number with increasing concentration of the bile salt. Bile salts can be purified by foam fractionation. Surface tension data for sodium cholate are in agreement with the above qualitative model of self-association. An isoextraction method is useful for estimating monomer activities. Results for sodium deoxycholate suggest little self-association in dilute solutions and a mildly cooperative self-association at higher concentrations. A comparative study of the interactions of the fluorescent probe, 2-p-toluidinyl-naphthalene-6-sulfonate, with sodium alkyl sulfates and sodium deoxycholate indicates that bile salts may differ significantly from classical micellar systems in their solubilization characteristics also. The evidence suggests strongly that a specific adduct formation with an optimum number of bile salt anions may be important in solubilization brought about by the rigidity and the complex shape of the bile salt anions.