Targeting NLRP3 Inflammasome Reduces Age-Related Experimental Alveolar Bone Loss.

The cause of chronic inflammatory periodontitis, which leads to the destruction of periodontal ligament and alveolar bone, is multifactorial. An increasing number of studies have shown the clinical significance of NLRP3-mediated low-grade inflammation in degenerative disorders, but its causal linkage to age-related periodontitis has not yet been elucidated. In this study, we investigated the involvement of the NLRP3 inflammasome and the therapeutic potential of NLRP3 inhibition in age-related alveolar bone loss by using in vivo and in vitro models. The poor quality of alveolar bones in aged mice was correlated with caspase-1 activation by macrophages and elevated levels of IL-1ß, which are mainly regulated by the NLRP3 inflammasome, in periodontal ligament and serum, respectively. Aged mice lacking Nlrp3 showed better bone mass than age-matched wild-type mice via a way that affects bone resorption rather than bone formation. In line with this finding, treatment with MCC950, a potent inhibitor of the NLRP3 inflammasome, significantly suppressed alveolar bone loss with reduced caspase-1 activation in aged mice but not in young mice. In addition, our in vitro studies showed that the addition of IL-1ß encourages RANKL-induced osteoclastogenesis from bone marrow-derived macrophages and that treatment with MCC950 significantly suppresses osteoclastic differentiation directly, irrelevant to the inhibition of IL-1ß production. Our results suggest that the NLRP3 inflammasome is a critical mediator in age-related alveolar bone loss and that targeting the NLRP3 inflammasome could be a novel option for controlling periodontal degenerative changes with age.

Observation of persistent species temperature separation in inertial confinement fusion mixtures.

The injection and mixing of contaminant mass into the fuel in inertial confinement fusion (ICF) implosions is a primary factor preventing ignition. ICF experiments have recently achieved an alpha-heating regime, in which fusion self-heating is the dominant source of yield, by reducing the susceptibility of implosions to instabilities that inject this mass. We report the results of unique separated reactants implosion experiments studying pre-mixed contaminant as well as detailed high-resolution three-dimensional simulations that are in good agreement with experiments. At conditions relevant to mixing regions in high-yield implosions, we observe persistent chunks of contaminant that do not achieve thermal equilibrium with the fuel throughout the burn phase. The assumption of thermal equilibrium is made in nearly all computational ICF modeling and methods used to infer levels of contaminant from experiments. We estimate that these methods may underestimate the amount of contaminant by a factor of two or more.

Jammed Limit of Bijel Structure Formation.

Over the past decade, methods to control microstructure in heterogeneous mixtures by arresting spinodal decomposition via the addition of colloidal particles have led to an entirely new class of bicontinuous materials known as bijels. Herein, we present a new model for the development of these materials that yields to both numerical and analytical evaluation. This model reveals that a single dimensionless parameter that captures both chemical and environmental variables dictates the dynamics and ultimate structure formed in bijels. We demonstrate that this parameter must fall within a fixed range in order for jamming to occur during spinodal decomposition, as well as show that known experimental trends for the characteristic domain sizes and time scales for formation are recovered by this model.

Arrest defective 1 regulates the oxidative stress response in human cells and mice by acetylating methionine sulfoxide reductase A.

Methionine sulfoxide reductase A (MSRA) protects proteins from oxidation, and also helps remove reactive oxygen species (ROS) by recovering antioxidant enzymes inactivated by oxidation. Although its functions have been investigated extensively, little is known about the mechanism by which MSRA is regulated. Arrest defective 1 (ARD1) is an enzyme that catalyzes not only N-terminal acetylation as a cotranslational modification but also lysine acetylation as a posttranslational modification. ARD1, which is expressed in most cell types, is believed to participate in diverse biological processes, but its roles are poorly understood. Given that MSRA was hunted in a yeast two-hybrid screen with ARD1 as the bait, we here investigated whether ARD1 is a novel regulator of MSRA. ARD1 was shown to interact with and acetylate MSRA in both cells and test tubes. It specifically acetylated the K49 residue of MSRA, and by doing so repressed the enzymatic function of MSRA. ARD1 increased cellular levels of ROS, carbonylated proteins and DNA breaks under oxidative stress. Moreover, it promoted cell death induced by pro-oxidants, which was attenuated in MSRA-deficient cells. When mice were exposed to hyperoxic conditions for 2 days, their livers and kidneys were injured and protein carbonylation was increased. The oxidative tissue injury was more severe in ARD1 transgenic mice than in their wild-type littermates. In conclusion, ARD1 has a crucial role in the cellular response to oxidative stress as a bona fide regulator of MSRA. ARD1 is a potential target for ameliorating oxidative injury or for potentiating ROS-producing anticancer agents.

Mosaic double aneuploidy (45,X/47,XX,+8) with aortic dissection.

Chromosomal aneuploidy is considerably frequent and may involve either autosomes or sex chromosomes. While double aneuploidy involving both autosomal and sex chromosomes is rare, several reports described the cases of sex chromosomal aneuploidies in combination with trisomy 21, such as Down-Klinefelter and Down-Turner syndrome. However, trisomy 8-Turner syndrome has been rarely described to date. Here we report a case of a 28-year-old female with mosaic trisomy 8-Turner syndrome. The patient was referred to our hospital for aortic dissection. On physical evaluation, features of her phenotype, which included short stature, webbed neck and cubitus valgus, suggested congenital anomalies such as Turner syndrome. Chest CT revealed aortic dissection with bicuspid aortic valve and coarctation. G-banding cytogenetic analysis of peripheral blood showed mosaicism with two cell lines (45,X[17]/47,XX,+8[33]). FISH analysis indicated that 15% of the cells were of monosomy X karyotype and 85% of the cells were with XX karyotype and trisomy 8 was detected only in XX cells. Though the patient exhibited clinical features of Turner syndrome, somatic stigmas present were not clearly distinguishable from those of trisomy 8, such as short stature, skeletal and cardiac abnormalities. Observations from most of the double aneuploidy cases indicated that the patient's phenotype was not necessarily in correlation to the ratio of autosomal and sex chromosomal aberrations. Mosaicism in trisomy 8-Turner syndrome was rarely documented and we believe this is the first reported case of mosaicism in trisomy 8-Turner syndrome presenting with aortic dissection and surviving into adulthood.

Effect of overweight/obesity on recovery after post-infectious glomerulonephritis.

BACKGROUND: Children with post-infectious glomerulonephritis (PIGN) rapidly recover and have excellent long-term outcomes. After encountering several overweight/obese children with persistent urinary abnormalities during recovery from PIGN, we conducted this retrospective study to determine if overweight/obese status prolonged time to resolution of renal abnormalities after PIGN. METHODS: Records of 20 children with PSGN evaluated between 1/98 and 12/05 were abstracted for demographics, clinical and laboratory data. Primary outcome measures were time to resolution of hypertension, proteinuria, microhematuria, and low complement C3. The effect of overweight/obese status on outcomes was determined using Kaplan Meier Survival and the log-rank test. RESULTS: The median age was 8 years; 30% were overweight/obese. At presentation, 17 (85%) were hypertensive, 10 (50%) had impaired glomerular filtration (GFR), and 18 (90%) had proteinuria. At last follow-up (median 2.8 months) 12% had hypertension, 55% had microhematuria, 5% had proteinuria but none had low GFR. Median time to normalize was: 30 days (GFR), 45 days (blood pressure), 6 weeks (C3) and 6 months (microhematuria). Log rank test showed that proteinuria-, hypertension-, and hematuria-free survival were all lower in children who were overweight/obese although none of the differences were statistically significant. Time to normalization of C3 was shorter in obese/ overweight children. CONCLUSION: In conclusion, overweight/obese children appear to have greater residual renal injury after PIGN. The earlier C3 normalization in overweight/ obese children may indicate that the adverse effect of weight on recovery is from hemodynamic rather than inflammatory factors. Close follow-up of overweight/obese children who develop PIGN is warranted to ensure optimal long-term outcomes.

Novel HLA-Cw*06 allele, Cw*0612, identified by sequence-based typing.

In this report, we describe the identification of an human leucocyte antigen-Cw*06 (HLA-Cw*06) nucleotide sequence variant, a new HLA-Cw*0612. The new allele was detected during routine HLA typing by high-resolution sequence-based typing. Allele Cw*0612 showed one nucleotide difference with Cw*0602 at codon 153 (GCG-->ACG) resulting in an amino acid change from alanine to threonine.

Novel HLA-Cw*01 allele, Cw*010203, identified by sequence-based typing*.

In this report, we describe the identification of an human leucocyte antigen-Cw*0102 (HLA-Cw*0102) nucleotide sequence variant, a new HLA-Cw*010203, in a case - control study by using sequence-based typing. Allele HLA-Cw*010203 showed one nucleotide difference with HLA-Cw*010201 by a silent substitution at codon 130 (CTG-->CTA).

Novel HLA-A*11 allele, A*1120, identified by sequence-based typing.

In this report, we describe the identification of a human leucocyte antigen-A*11 (HLA-A*11) nucleotide sequence variant, a new HLA-A*1120 by using sequence-based typing (SBT). The new allele was detected during routine HLA typing by high-resolution SBT. Allele A*1120 showed one nucleotide difference with A*110101 at codon 152 (GCG-->GAG) resulting in an amino acid change from alanine to glutamate. Residue 152 is located on alpha(2)-helix of HLA class I molecule and involved in peptide binding by constructing E pocket of peptide-binding groove, implying that the change of the residue 152 would affect the binding affinity of peptides to A*1120 allele.

Novel HLA-A*31 allele, A*3111 identified by sequence-based typing.

In this report, we describe the identification of an HLA-A*31 nucleotide sequence variant, a new HLA-A*3111, in three members of a Korean family by using sequence-based typing (SBT). The new allele was detected during routine HLA typing by high-resolution SBT. Allele A*3111 showed one nucleotide difference with A*310102 at codon 165 (GTG-->CTG) resulting in an amino acid change from valine to leucine (V165L). Serologic reactivity was shorter than normally expected.

Arabidopsis thaliana ferrochelatase-I and -II are not imported into Arabidopsis mitochondria.

Using in vitro import assays into purified mitochondria and chloroplasts we found that Arabidopsis ferrochelatase-I and ferrochelatase-II were not imported into mitochondria purified from Arabidopsis (or several other plants) but were imported into pea leaf chloroplasts. Other dual targeted proteins could be imported into purified mitochondria from Arabidopsis. As only two ferrochelatase genes are present in the completed Arabidopsis genome, the presence of ferrochelatase activity in plant mitochondria needs to be re-evaluated. Previous reports of Arabidopsis ferrochelatase-I import into pea mitochondria are due to the fact that pea leaf (and root) mitochondria appear to import a variety, but not all chloroplast proteins. Thus pea mitochondria are not a suitable system to either study dual targeting, or to distinguish between isozymes present in mitochondria and chloroplasts.

The repressive activity of hepatitis C virus core protein on the transcription of p21(waf1) is regulated by protein kinase A-mediated phosphorylation.

Hepatitis C virus (HCV) core protein is known to repress the transcription of p21(waf1) directly in a p53-independent manner. In this study, the region of HCV core protein responsible for the transcriptional repression of p21 promoter was determined. N-terminal half of core protein almost completely lost the ability to repress p21 promoter, indicating that the domain required for the majority of p21 repression is located between amino acid positions 84 and 191. The trans-repression activity of HCV core mutant S99L on p21 gene expression was similar to that of wild type core protein whereas mutation of the 116th amino acid Ser into either Ile or Ala completely abolished the repressive ability of HCV core protein. In addition, the trans-repression activity of HCV core mutant S116D was similar to that of wild type core protein, suggesting that an acidic aspartate residue can mimic the effect of phosphorylation. When treated with a protein kinase A (PKA) inhibitor, H-89, the inhibitory activity of wild-type HCV core protein was dose-dependently decreased and was completely lost at the concentration of 5 microM. On the contrary, the repression activity of HCV core protein was increased by treatment with a PKA activator, dibutyryl-cAMP, indicating that the p21 repressive activity of HCV core protein is regulated by phosphorylation at S-116 by protein PKA

Roles of phosphatidylinositol 3-kinase and Rac in the nuclear signaling by tumor necrosis factor-alpha in rat-2 fibroblasts.

We investigated the extent to which phosphatidylinositol 3-kinase (PI 3-kinase) and Rac, a member of the Rho family of small GTPases, are involved in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha leading to activation of c-fos serum response element (SRE) and c-Jun amino-terminal kinase (JNK) in Rat-2 fibroblasts. Inhibition of PI 3-kinase by LY294002 or wortmannin, two specific PI 3-kinase antagonists, or co-transfection with a dominant negative mutant of PI 3-kinase dose-dependently blocked stimulation of c-fos SRE by TNF-alpha. Similarly, LY294002 significantly diminished TNF-alpha-induced activation of JNK, suggesting that nuclear signaling triggered by TNF-alpha is dependent on PI 3-kinase-mediated activation of both c-fos SRE and JNK. We also found nuclear signaling by TNF-alpha to be Rac-dependent, as demonstrated by the inhibitory effect of transient co-transfection with a dominant negative Rac mutant, RacN17. Our findings suggest that Rac is situated downstream of PI 3-kinase in the TNF-alpha signaling pathway to the nucleus, and we conclude that PI 3-kinase and Rac each plays a pivotal role in the nuclear signaling cascade triggered by TNF-alpha.

The effects of ultrarapid freezing on meiotic and mitotic spindles of mouse oocytes and embryos.

Preovulatory mouse oocytes and 2-cell embryos were frozen with dimethyl sulfoxide and propanediol by an ultrarapid method. The survival of frozen oocytes was low (33-34%) compared to that of 2-cell embryos (78-79%) with either cryoprotectant. Development to blastocysts after postthaw culture was about 7-15% for oocytes and 79-80% for the embryos. Ultrarapid freezing preserves cell structure quite well as revealed by electron microscopy, but meiotic oocytes and late 2-cell embryos undergoing mitosis showed evidence of spindle disorganization involving loss or clumping of microtubules resulting in some scattering of chromosomes. Embryos developed from frozen eggs showed clear evidence of micronuclear formation and incomplete incorporation of chromosomal material into main nuclei. These experiments confirm our observations on freezing of human oocytes and show that spindle microtubules are sensitive to freeze-thawing and that cryopreservation could cause chromosomal aberrations during early development. A cautious approach to the introduction of oocyte freezing in human in vitro fertilization (IVF) programs is advocated.

The use of amniotic fluid and serum with propanediol in freezing of murine 2-cell embryos.

Human and mouse embryos have been cultured in amniotic fluid (AF). Human AF and human serum (HS) are used in the freeze-thaw of 2-cell mouse embryos. Two hundred seventy-five 2-cell embryos were collected into phosphate-buffered saline with 20% HS and 20% AF and into 100% HS and AF. The embryos were cooled with propanediol as cryoprotectant at a controlled rate. After thaw, they were cultured in T6 with 3 mg/ml bovine serum albumin. Blastocyst formation post-thaw was 56/79, 44/70, 51/61, and 56/79 of intact embryos from 20% HS, 20% AF, pure HS, and pure AF (NS). But blastocyst hatching was better from embryos frozen in pure HS (22/61, compared with 16/79 for 20% HS; P less than 0.05). Hence there is no advantage in using AF in freeze-thaw, but pure HS may be of use.