Smooth muscle differentiation of coronary intima in autopsy tissues after sirolimus-eluting stent implantation.

BACKGROUND: In coronary atherosclerotic disease, the proliferation of intimal smooth muscle cells (SMCs) is regarded as beneficial with respect to stable and unstable plaques, but is thought detrimental in discussions on coronary stent restenosis. To resolve this discrepancy, we focused on the quality, not quantity, of intimal SMCs in coronary atherosclerotic disease. METHODS: Autopsied coronary artery specimens from seven patients implanted with bare metal stents (BMS), three with paclitaxel-eluting stents (PES), and 10 with sirolimus (rapamycin)-eluting stents (SES) were immunostained for SMC markers. Cultured human coronary artery SMCs were also treated with sirolimus and paclitaxel. RESULTS: Intimal SMC differentiation, estimated by the ratio of h-caldesmon+ cells to α-smooth muscle actin+ (α-SMA+) cells, was significantly increased whereas dedifferentiation, estimated from the ratio of fibroblast activation protein alpha (FAPα)+ cells to α-SMA+ cells, was significantly decreased, in tissues of SES compared with BMS cases. No difference in the degree of differentiation was found between PES and BMS cases or between the three groups in nonstented arteries used as controls. Correlation analyses for each field of view revealed a significant positive correlation between h-caldesmon and calponin staining but significant negative correlations with FAPα staining in α-SMA+ cells. Cultured SMCs were shorter (dedifferentiated) and showed an increased FAPα/α-SMA protein when treated with paclitaxel, whereas they became elongated (differentiated) and showed increased calponin/α-SMA proteins with sirolimus. CONCLUSIONS: The SMCs of the coronary intima may differentiate after SES implantation. SMC differentiation may explain both the plaque stabilization and reduced risk of reintervention associated with SES.

An improved macromolecular crowding sensor CRONOS for detection of crowding changes in membrane-less organelles under stressed conditions.

Membrane-less organelles (MLOs) formed by liquid-liquid phase separation (LLPS) play pivotal roles in biological processes. During LLPS, proteins and nucleotides are extremely condensed, resulting in changes in their conformation and biological functions. Disturbed LLPS homeostasis in MLOs is thought to associate with fatal diseases such as amyotrophic lateral sclerosis. Therefore, it is important to detect changes in the degree of crowding in MLOs. However, it has not been investigated well due to the lack of an appropriate method. To address this, we developed a genetically encoded macromolecular crowding sensor CRONOS (crowding sensor with mNeonGreen and mScarlet-I) that senses the degree of macromolecular crowding in MLOs using a fluorescence resonance energy transfer (FRET) system. CRONOS is a bright biosensor with a wide dynamic range and successfully detects changes in the macromolecular volume fraction in solution. By fusing to the scaffold protein of each MLO, we delivered CRONOS to MLO of interest and detected previously undescribed differences in the degree of crowding in each MLO. CRONOS also detected changes in the degree of macromolecular crowding in nucleolus induced by environmental stress or inhibition of transcription. These findings suggest that CRONOS can be a useful tool for the determination of molecular crowding and detection of pathological changes in MLOs in live cells.

Phase separation and toxicity of C9orf72 poly(PR) depends on alternate distribution of arginine.

Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid-liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein-protein interactions.

Establishment of chemically oligomerizable TAR DNA-binding protein-43 which mimics amyotrophic lateral sclerosis pathology in mammalian cells.

One of the pathological hallmarks of amyotrophic lateral sclerosis (ALS) is mislocalized, cytosolic aggregation of TAR DNA-Binding Protein-43 (TDP-43). Not only TDP-43 per se is a causative gene of ALS but also mislocalization and aggregation of TDP-43 seems to be a common pathological change in both sporadic and familial ALS. The mechanism how nuclear TDP-43 transforms into cytosolic aggregates remains elusive, but recent studies using optogenetics have proposed that aberrant liquid-liquid phase separation (LLPS) of TDP-43 links to the aggregation process, leading to cytosolic distribution. Although LLPS plays an important role in the aggregate formation, there are still several technical problems in the optogenetic technique to be solved to progress further in vivo study. Here we report a chemically oligomerizable TDP-43 system. Oligomerization of TDP-43 was achieved by a small compound AP20187, and oligomerized TDP-43 underwent aggregate formation, followed by cytosolic mislocalization and induction of cell toxicity. The mislocalized TDP-43 co-aggregated with wt-TDP-43, Fused-in-sarcoma (FUS), TIA1 and sequestosome 1 (SQSTM1)/p62, mimicking ALS pathology. The chemically oligomerizable TDP-43 also revealed the roles of the N-terminal domain, RNA-recognition motif, nuclear export signal and low complexity domain in the aggregate formation and mislocalization of TDP-43. The aggregate-prone properties of TDP-43 were enhanced by a familial ALS-causative mutation. In conclusion, the chemically oligomerizable TDP-43 system could be useful to study the mechanisms underlying the droplet-aggregation phase transition and cytosolic mislocalization of TDP-43 in ALS and further study in vivo.

Characterization of membrane penetration and cytotoxicity of C9orf72-encoding arginine-rich dipeptides.

Cell-penetrating peptides (CPPs) including arginine-rich peptides are attracting a lot of attention due to their potential as a novel intracellular drug delivery tool without substantial toxicity. On the other hand, disease-associated arginine-rich CPPs, such as poly-PR and poly-GR translated from C9orf72 gene, also efficiently enter neuronal cells and then kill them. Although both non-harmful CPPs and harmful poly-PR/GR penetrate the plasma membrane using same arginine residues, little is known about the factors which determine the toxicity of the pathogenic CPPs. Here, we show that poly-PR and poly-GR, but not other Arg-rich CPPs, specifically distributed to nucleolus via interaction with RNA. Importantly, C9orf72-dipeptides, but not other Arg-rich CPPs, caused inhibition of protein translation and cell death. Raising extracellular pH enhanced the cell penetration of poly-PR. The repeat number of (PR) affected the secondary structure and determined the intracellular delivery rate and neurotoxicity, and enforced intracellular delivery of non-penetrating short poly-PR peptide caused cell death, suggesting that modulation of extracellular environment to inhibit the uptake of Arg-rich dipeptides might be a drug target against poly-PR/GR-mediated neurotoxicity.

Early predictors of status epilepticus-associated mortality and morbidity in children.

BACKGROUND: Early predictors of status epilepticus (SE)-associated mortality and morbidity have not been systematically studied in children, considerably impeding the identification of patients at risk. OBJECTIVES: To determine reliable early predictors of SE-associated mortality and morbidity and identify the etiology of SE-associated sequelae in Japanese children. METHODS: We conducted a prospective multicenter study of clinical findings and initial laboratory data acquired at SE onset, and assessed outcomes at the last follow-up examination. In-hospital death during the acute period and neurological sequelae were classified as poor outcomes. RESULTS: Of the 201 children who experienced their first SE episode, 16 exhibited poor outcome that was most commonly associated with acute encephalopathy. Univariate analysis revealed that the following were associated with poor outcomes: young age (⩽24 months); seizure duration >90 min; seizure intractability (failure of the second anticonvulsive drug); biphasic seizures; abnormal blood glucose levels (<61 or >250 mg/dL); serum aspartate aminotransferase (AST) ⩾56 U/L; and C-reactive protein (CRP) levels >2.00 mg/dL. Multivariate analysis revealed that young age, seizure intractability, abnormal blood glucose levels, and elevated AST and CRP levels were statistically significant. CONCLUSIONS: Young age and seizure intractability were highly predictive of poor outcomes in pediatric SE. Moreover, abnormal blood glucose levels and elevated AST and CRP levels were predictors that might be closely associated with the etiology, especially acute encephalopathy and severe bacterial infection (sepsis and meningitis) in Japanese children.

[Influenza-associated encephalopathy with onset of prolonged convulsion: a case report].

A 1-year-old girl with influenza-associated encephalopathy initially exhibited prolonged febrile convulsions and subsequent drowsiness. She became unconsciousness and experienced a cluster of seizures 4 days later. Diffusion-weighted magnetic resonance imaging (DWI) showed high signal intensity in the bilateral frontal white matter. This signal change migrated to the bifrontal cortical areas and the caudate nuclei within 10 days, when T2 elongation appeared over the gray and white matter of frontal lobes. Choreoathetosis and oculogyric crisis were transiently noted at this period. Frontal lobe signs, including the forded mouth opening response, after diminution of these movement disorders. The DWI signal change subsequently resolved and frontal cortical atrophy appeared thereafter. Levels of inflammatory cytokines in the cerebrospinal fluid were normal during the acute phase of clinical course. The biphasic clinical course with initial prolonged seizure, involvement of the frontal lobes, and the progression of signal change on DWI from white to gray matter, meets the characteristics of "status epilepticus-type acute encephalopathy" suggested by Shiomi et al. Although pentobarbital infusion, steroid pulse therapy and mild hypothermia did not show any apparent effects on the clinical course of this patient, further trial of these therapies may be warranted for the treatment of this type of encephalopathy.

Corneal elasticity as a measure of intra-ocular pressure: a controlled clinical examination.

PURPOSE: To determine the clinical efficacy of corneal elasticity (CE) measurements as an index of intra-ocular pressure(IOP). The considerable body of evidence linking elevated intra-ocular pressure (IOP) to glaucomatous conditions, points to the importance of the accurate and reliable measurement of IOP for diagnosis, prevention and post treatment assessments. Recently an applanation resonator sensor (ARS) for measuring IOP via CE has been proposed as an alternative to the extant Goldmann applanation tonometry (GAT). The present study examines the validity of the measure as an index of IOP in glaucomatous patients. METHODS: A structured sample of glaucomatous patients and orthogonally matched normal control subjects were assessed by the GAT and ARS methods in the research and outpatient treatment laboratory of the university Ophthalmology Department. The patient sample consisted of individuals with IOP of 16 mmHg and above and with evidence of clinically significant optic nerve damage. RESULTS: Significant correlations between GAT-IOP and ARS frequency shifts (Delta fs) for both clinical (r=.88, p<.001, n=16) and normal subjects (r=.60, p<.01, n=16) were obtained. CONCLUSIONS: The results suggest that ARS could be a clinical alternative to the extant GAT method in assessing IOP. In order to further examine the clinical efficacy of ARS-Delta fs as an accurate index of IOP, tests of a significant correlation between ARS-Delta fs and manometrically established vitreous IOP are now needed in normal and glaucomatous subjects.