Seizure Outcomes and Predictors of Recurrent Post-Stroke Seizure: A Retrospective Observational Cohort Study

Background

Seizure is a common complication after stroke (termed “post-stroke seizure,” PSS). Although many studies have assessed outcomes and risk factors of PSS, no reliable predictors are currently available to determine PSS recurrence. We compared baseline clinical characteristics and post-stroke treatment regimens between recurrent and non-recurrent PSS patients to identify factors predictive of recurrence.

Methods

Consecutive PSS patients admitted to our stroke center between January 2011 and July 2013 were monitored until February 2014 (median 357 days; IQR, 160–552) and retrospectively evaluated for baseline clinical characteristics and PSS recurrence. Cumulative recurrence rates at 90, 180, and 360 days post-stroke were estimated by Kaplan—Meier analysis. Independent predictors of recurrent PSS were identified by Cox proportional-hazards analysis.

Results

A total of 104 patients (71 men; mean age, 72.1 ± 11.2 years) were analyzed. PSS recurred in 31 patients (30%) during the follow-up. Factors significantly associated with PSS recurrence by log-rank analysis included previous PSS, valproic acid (VPA) monotherapy, polytherapy with antiepileptic drugs (AEDs), frontal cortical lesion, and higher modified Rankin Scale score at discharge (all p < 0.05). Independent predictors of recurrent PSS were age <74 years (HR 2.38, 95% CI 1.02–5.90), VPA monotherapy (HR 3.86, 95% CI 1.30–12.62), and convulsions on admission (HR 3.87, 95% CI 1.35–12.76).

Conclusions

Approximately one-third of PSS patients experienced seizure recurrence within one year. The predictors of recurrent PSS were younger age, presence of convulsions and VPA monotherapy. Our findings should be interpreted cautiously in countries where monotherapy with second-generation AEDs has been approved because this study was conducted while second-generation AEDs had not been officially approved for monotherapy in Japan.     

Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from Streptomyces Species

β-D-galactofuranose (Galf) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Galf-ase) gene has been identified in any organism. To search for a Galf-ase gene we screened soil samples and discovered a strain, identified as a Streptomyces species by the 16S ribosomal RNA gene analysis, that exhibits Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Galf-ases. Using recombinant proteins expressed in Escherichia coli, we found that three out of four candidates displayed the activity of not only Galf-ase but also α-L-arabinofuranosidase (Araf-ase), whereas the other one showed only the Galf-ase activity. This novel Galf-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate pNP-β-D-Galf. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus Aspergillus fumigatus, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Galf-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Galf-specific Galf-ases widely exist in microorganisms.     

Cell-free identification of novel N-myristoylated proteins from complementary DNA resources using bioorthogonal myristic acid analogues

To establish a non-radioactive, cell-free detection system for protein N-myristoylation, metabolic labeling in a cell-free protein synthesis system using bioorthogonal myristic acid analogues was performed. After Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) with a biotin tag, the tagged proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and blotted on a polyvinylidene fluoride (PVDF) membrane, and then protein N-myristoylation was detected by enhanced chemiluminescence (ECL) using horseradish peroxidase (HRP)-conjugated streptavidin. The results showed that metabolic labeling in an insect cell-free protein synthesis system using an azide analogue of myristic acid followed by CuAAC with alkynyl biotin was the most effective strategy for cell-free detection of protein N-myristoylation. To determine whether the newly developed detection method can be applied for the detection of novel N-myristoylated proteins from complementary DNA (cDNA) resources, four candidate cDNA clones were selected from a human cDNA resource and their susceptibility to protein N-myristoylation was evaluated using the newly developed strategy. As a result, the products of three cDNA clones were found to be novel N-myristoylated protein, and myristoylation-dependent specific intracellular localization was observed for two novel N-myristoylated proteins. Thus, the metabolic labeling in an insect cell-free protein synthesis system using bioorthogonal azide analogue of myristic acid was an effective strategy to identify novel N-myristoylated proteins from cDNA resources.     

Secretion of inflammatory factors from chondrocytes by layilin signaling

Layilin (LAYN) is thought to be involved in reorganization of cytoskeleton structures, interacting with merlin, radixin, and talin. Also, LAYN is known to be one of the receptors for hyaluronic acid (HA).     

Characterization of abdominal appendages in the sawfly, Athalia rosae (Hymenoptera), by morphological and gene expression analyses

Larvae of the sawfly, Athalia rosae, have remarkable abdominal prolegs. We analyzed the morphogenesis of appendages and the expression of decapentaplegic and Distal-less genes during embryonic development to characterize the origin of prolegs. Proleg primordia in abdominal segments A1–A9 appeared shortly after the inner lobes (endites) of gnathal appendages were formed. These were located on the ventral plates, medioventral to the appendages of the other segments in light of serial homology. Nothing was seen where the main axis of the appendage should develop in abdominal segments. The primordia in A1 and A9 disappeared before larval hatching. Anal prolegs appeared separate from cerci, the main axes of appendages, which were formed temporarily in A11. The expression of decapentaplegic, which reflects the primary determination of appendages, was detected in the lateral juxtaposition with the prolegs. Distal-less was expressed in the main axes of appendages, protruding endites and the cerci, but not in prolegs and anal prolegs or the gnathal endites which do not protrude. These findings suggest a possibility that the abdominal and anal prolegs of A. rosae are outgrowths of ventral plates which derived from coxopodal elements, but not main axes of appendages.     

Purification,Biochemical Characterization,and Cloning of Phospholipase D from <Emphasis Type="Italic">Streptomyces racemochromogenes</Emphasis> Strain 10-3

We previously isolated Streptomyces racemochromogenes strain 10-3, which produces a phospholipase D (PLD) with high transphosphatidylation activity. Here, we purified and cloned the PLD (PLD103) from the strain. PLD103 exerted the highest hydrolytic activity at a slightly alkaline pH, which is in contrast to the majority of known Streptomyces PLDs that have a slightly acidic optimum pH. PLD103 shares only 71–76% amino acid sequence identity with other Streptomyces PLDs that have a slightly acidic optimum pH; thus, the diversity in the primary structure might explain the discrepancy observed in the optimum pH. The purified PLD displayed high transphosphatidylation activity in the presence of glycerol, l-serine, and 2-aminoethanol hydrochloride with a conversion rate of 82–97% in a simple one-phase system, which was comparable to the rate of other Streptomyces PLDs in a complicated biphasic system.     

Protective effect of nicotinamide against poly(ADP-ribose) polymerase-1-mediated astrocyte death depends on its transporter-mediated uptake

AimPoly(ADP-ribose) polymerase-1 (PARP-1) is a DNA repair enzyme, and its excessive activation, following ischemia, trauma, etc., depletes cellular nicotinamide adenine dinucleotide (NAD⁺) as a substrate and eventually leads to brain cell death. Nicotinamide, an NAD⁺ precursor and a PARP-1 inhibitor, is known to prevent PARP-1-triggered cell death, but there is no available information on the mechanisms involved in its transport. Here we clarified the transport characteristics of nicotinamide in primary cultured mouse astrocytes.Main methodsUptake characteristics of [¹⁴C]nicotinamide were assessed by a conventional method with primary cultured mouse astrocytes. Cell viability and PARP-1 activity were determined with intracellular LDH activity and immunocytochemical detection of PAR accumulation, respectively.Key findingsPARP-1 activation was induced by treatment of astrocytes with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent. MNNG-triggered astrocyte death and PAR accumulation were completely inhibited by treatment with nicotinamide as with DPQ (3,4-dihydro-5-(4-(1-piperidinyl)butoxy)-1(2H)-isoquinolinone), a second generation PARP inhibitor. The uptake of [¹⁴C]nicotinamide was time-, temperature-, concentration- and pH-dependent, and was inhibited and stimulated by co- and pre-treatment with N-methylnicotinamide, a representative substrate of an organic cation transport system, respectively. Co-treatment of astrocytes with nicotinamide and N-methylnicotinamide resulted in a decrease in PAR accumulation and absolute prevention of cell death.SignificanceThese findings suggest that nicotinamide has a protective effect against PARP-1-induced astrocyte death and that its transporter-mediated uptake, which is extracellular pH-sensitive and common to N-methylnicotinamide, is critical for prevention of PARP-1-triggered cell death.