Vascular Ehlers-Danlos Syndrome Mutations in Type III Collagen Differently Stall the Triple Helical Folding

Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)₇ with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)₇-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.     

Isolation of a cDNA clone encoding mouse 3-hydroxyacyl CoA dehydrogenase

Rae-38, a cDNA clone isolated from mouse embryonal carcinoma F9 cells, was sequenced, and the deduced RAE-38 protein showed about 86% homology to pig 3-hydroxyacyl CoA dehydrogenase (HCDH; EC 1.1.1.35). This clone can be used to elucidate the regulatory mechanism of HCDH gene expression in mammals.     

A Novel Acylaminoimidazole Derivative,WN1316, Alleviates Disease Progression via Suppression of Glial Inflammation in ALS Mouse Model

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron degenerative disease. Given that oxidative stress and resulting chronic neuronal inflammation are thought to be central pathogenic, anti-oxidative agents and modulators of neuronal inflammation could be potential therapies for ALS. We report here that the novel small molecular compound, 2-[mesityl(methyl)amino]-N-[4-(pyridin-2-yl)-1H-imidazol-2-yl] acetamide trihydrochloride (WN1316) selectively suppresses oxidative stress-induced cell death and neuronal inflammation in the late-stage ALS mice. WN1316 has high blood-brain-barrier permeability and water solubility, and boosts both neuronal apoptosis inhibitory protein (NAIP) and NF-E2-related factor 2 (Nrf2) which governed glutathione (GSH)-related anti-oxidation pathway protecting motor neurons against oxidative injuries. Post-onset oral administration of low dose (1–100 µg/kg/day) WN1316 in ALS(SOD1^H46R) and ALS(SOD1^G93A) mice resulted in sustained improved motor function and post onset survival rate. Immunohistochemical analysis revealed less DNA oxidative damage and motor neuronal inflammation as well as repression of both microgliosis and astrocytosis, concomitant down regulation of interleukin-1β and inducible nitric oxide synthase, and preservation of the motoneurons in anterior horn of lumbar spinal cord and skeletal muscle (quadriceps femoris). Thus, WN1316 would be a novel therapeutic agent for ALS.     

Influence of surface anesthesia on the pressure pain threshold measured with different-sized probes

Transcutaneous pressure with pressure probes of arbitrary diameters have been commonly used for measuring the threshold and magnitude of muscle pain, yet this procedure lacks scientific validation. To examine the valid probe dimensions, we conducted physiological experiments using 34 human subjects. Pin-prick pain, pressure pain threshold (PPT) to pressure probes of various diameters, heat pain threshold, and electrical pain threshold of deep tissues were measured before and after application of surface lidocaine anesthesia to the skin surface over the brachioradial muscle in a double-blinded manner. The anesthesia neither affected PPT with larger probes (diameters: 1.6 and 15?mm) nor increased electric pain threshold of deep structures, whereas it diminished pain count in pin-prick test and PPT with a 1.0?mm diameter probe, suggesting that mechanical pain thresholds measured with 1.6 and 15?mm probes reflect the pain threshold of deep tissues, possibly muscle. Pain thresholds to heat did not change after application of the anesthesia. These results suggest that larger pressure probes can give a better estimation of muscular pain threshold.     

Model studies for isolation of G-quadruplex-forming DNA sequences through a pull-down strategy with macrocyclic polyoxazole

G-quadruplexes (G4s) are non-B DNA structures present in guanine-rich regions of gene regulatory areas, promoters and CpG islands, but their occurrence and functions remain incompletely understood. Thus, methodology to identify G4 sequences is needed. Here, we describe the synthesis of a novel cyclic hepta-oxazole compound, L1Bio-7OTD (1), bearing a biotin affinity-tag as a tool to pull down G4 structures from mixtures of G4-forming and non G4-forming DNA sequences. We confirmed that it could pull down G4s associated with telomeres, bcl-2 gene, and c-kit gene.     

Cave insects with sex‐reversed genitalia had their most recent common ancestor in West Gondwana (Psocodea: Prionoglarididae: Speleketorinae)

The divergence date and ancestral distributional area of the psocid subfamily Speleketorinae, which includes taxa with reversed genitalia (female penis and male vagina of Afrotrogla and Neotrogla, tribe Sensitibillini), were estimated. The most basal divergence of the subfamily (between the North American Speleketor and the tribe Sensitibillini) was estimated to have occurred according to the separation between the North American continent and Gondwana, ca. 175 Ma. The most basal divergence of Sensitibillini (between African Afrotrogla + Sensitibilla and Brazilian Neotrogla) was estimated to have occurred according to the split of West Gondwana (separation between the African and South American continents), ca. 127 Ma. The biome of the ancestral distributional area of Sensitibillini (inland of West Gondwana) is believed to be arid to semi‐arid, which might strengthen the reversed sexual selection and then facilitate the origin of preadaptive features related to the evolution of a female penis. All extant Sensitibillini species inhabit carbonatic caves, but geological evidence suggested independent shifts of these genera to the carbonatic cave habitat in the Tertiary/Quaternary.     

TAE226, a Bis-Anilino Pyrimidine Compound,Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells

TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation.     

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.