The Chronological Characteristics of SOD1 Activity and Inflammatory Response in the Hippocampi of STZ-Induced Type 1 Diabetic Rats

Because it appears that oxidative stress and inflammation are implicated with disease pathogenesis in the diabetic brain, many researchers have used streptozotocin (STZ)-induced diabetic animals to study superoxide production and the effects of superoxide scavengers like Cu,Zn-superoxide dismutase (SOD1). However, many studies have been conducted without considering temporal changes after STZ injection. Interestingly, though SOD activities were not significantly different among the groups, SOD1 and 4-hydroxy-2-nonenal (4-HNE) immunoreactivities were significantly enhanced at 3 weeks after an STZ injection (STZ3w) versus only marginal levels in sham controls, whereas microglial activity was remarkably reduced in injected rats at this time. However, SOD1 immunoreactivity and microglial activities were only at the sham level at STZ4w. The present study provides important information concerning cell damage by ROS generated by STZ. Microglial response was found to be inactivated at STZ3w and neuronal cells (NeuN) showed a non-significant tendency to be reduced in number at STZ4w except in the dentate gyrus. We speculated that the above oxidative stress-related events should be accomplished at STZ3w in the brains of STZ-induced diabetes animal models. Therefore, the aim of the present study was to investigate chronological changes in SOD1 immunoreactivity associated with lipid peroxidation and inflammatory responses in the hippocampi of STZ-induced type I diabetic rats.     

Time-course alterations of Toll-like receptor 4 and NF-κB p65, and their co-expression in the gerbil hippocampal CA1 region after transient cerebral ischemia

Innate immune system is very important to modulate the host defense against a large variety of pathogens. Toll-like receptors (TLRs) play a key role in controlling innate immune response. Among TLRs, TLR4 is a specific receptor for lipopolysaccharide and associated with the release of pro-inflammatory cytokines. In the present study, we investigated ischemia-related changes of TLR4 immunoreactivity and its protein level, and nuclear factor κB (NF-κB) p65 immunoreactivity regarding inflammatory responses in the hippocampal CA1 region after 5 min of transient cerebral ischemia to identify the correlation between transient ischemia and inflammation. In the sham-operated group, TLR4 immunoreactivity was easily detected in pyramidal neurons of the hippocampal CA1 region (CA1). TLR4 immunoreactivity in pyramidal neurons was distinctively decreased after ischemia/reperfusion (I/R); instead, based on double immunofluorescence study, TLR4 immunoreactivity was expressed in non-pyramidal neurons and astrocytes from 2 days postischemia. In addition, TLR4 protein level was lowest at 1 day postischemia and highest 4 days after I/R. On the other hand, NF-κB p65 immunoreactivity was not detected in the CA1 of the sham-operated group, and NF-κB p65 immunoreactivity was not observed until 1 day after I/R. However, NF-κB p65 immunoreactivity began to be expressed in astrocytes at 2 days postischemia, and the immunoreactivity was strong 4 days postischemia. Our results indicate that TLR4 and NF-κB p65 immunoreactivity are changed in CA1 pyramidal neurons and newly expressed in astrocytes, not in microglia, in the CA1 region after transient cerebral ischemia.     

Time-Course of Changes in Phosphorylated CREB in Neuroblasts and BDNF in the Mouse Dentate Gyrus at Early Postnatal Stages

Cyclic AMP (cAMP) response element-binding protein (CREB) is involved in memory, learning, and synaptic transmission. In this study, we observed changes of phosphorylated CREB (pCREB) immunoreactivity and its protein levels as well as brain-derived neurotrophic factor (BDNF) levels in the hippocampal dentate gyrus at postnatal (P) 1, 7, 14, and 21 in mice. In addition, we also investigated pCREB expression in doublecortin (DCX, a marker for neuronal progenitors) immunoreactive neuroblasts at P21. pCREB immunoreaction at P1 was detected in most of cells in the dentate gyrus, thereafter pCREB immunoreactivity was decreased in all the layers of the dentate gyrus with time, however, strong pCREB immunoreactivity was shown in cells confined to the subgranular zone of the dentate gyrus at P21. In this group, many pCREB immunoreactive cells were co-localized with DCX immunoreactive neuroblasts. In addition, pCREB protein levels were decreased with age, showing that their levels were very low at P21, while BDNF protein levels were increased with age. These results suggest that pCREB may play important roles in functional maturity of granule cells in mice.     

Systematic Study on Genetic and Epimutational Profile of a Cohort of Amsterdam Criteria-Defined Lynch Syndrome in Singapore

Background

Germline defects of mismatch repair (MMR) genes underlie Lynch Syndrome (LS). We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region.

Methods

Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI) was assessed with five mononucleotide markers and immunohistochemical staining (IHC) was also performed.

Results

Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8%) families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T) was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations.

Conclusions

Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.     

Antifilarial activity of intravenous suramin and oral diethylcarbamazine citrate on subperiodic Brugia malayi in the leaf-monkey, Presbytis cristata

The known filaricides, suramin and diethylcarbamazine citrate, were tested against subperiodic Brugia malayi infection in the leaf-monkey, Presbytis cristata. As expected, intravenous suramin at 10 mg/kg daily x 5 days or 17 mg/kg weekly x 5 weeks, did not show any microfilaricidal activity, but substantially reduced the recovery of live adult worms to 50.6% and 13.6% of controls respectively. Oral diethylcarbamazine citrate at 6 mg/kg daily x 6 or 10 days reduced final microfilarial counts to 30% of initial counts four weeks post-treatment and adult worm recovery was reduced to 4.5% and 0% of controls respectively. Although the antifilarial activity of these drugs against the infection in this non-human primate model appears to be similar to that seen in man, these results have to be confirmed using larger groups of animals.     

A rapid bio‐optical sensor for diagnosing Q fever in clinical specimens

Recent zoonotic outbreaks, such as Zika, Middle East respiratory syndrome and Ebola, have highlighted the need for rapid and accurate diagnostic assays that can be used to aid pathogen control. Q fever is a zoonotic disease caused by the transmission of Coxiella burnetii that can cause serious illness in humans through aerosols and is considered a potential bioterrorism agent. However, the existing assays are not suitable for the detection of this pathogen due to its low levels in real samples. We here describe a rapid bio‐optical sensor for the accurate detection of Q fever and validate its clinical utility. By combining a bio‐optical sensor, that transduces the presence of the target DNA based on binding‐induced changes in the refractive index on the waveguide surface in a label‐free and real‐time manner, with isothermal DNA amplification, this new diagnostic tool offers a rapid (<20 min), 1‐step DNA amplification/detection method. We confirmed the clinical sensitivity (>90%) of the bio‐optical sensor by detecting C. burnetii in 11 formalin‐fixed, paraffin‐embedded liver biopsy samples from acute Q fever hepatitis patients and in 16 blood plasma samples from patients in which Q fever is the cause of fever of unknown origin.      

Development of novel tetra- and trinucleotide microsatellite markers for giant grouper <Emphasis Type="Italic">Epinephelus lanceolatus</Emphasis> using 454 pyrosequencing

Giant grouper (Epinephelus lanceolatus) is a commercially important species, but its wild population has recently been classified as vulnerable. This species has significant potential for use in aquaculture, though a greater understanding of population genetics is necessary for selective breeding programs to minimize kinship for genetically healthy individuals. High-throughput pyrosequencing of genomic DNA was used to identify and characterize novel tetra- and trinucleotide microsatellite markers in giant grouper from Sabah, Malaysia. In total, of 62,763 sequences containing simple sequence repeats (SSRs) were obtained, and 78 SSR loci were selected to possibly contain tetra- and trinucleotide repeats. Of these loci, 16 had tetra- and 8 had trinucleotide repeats, all of which exhibited polymorphisms within easily genotyped regions. A total of 143 alleles were identified with an average of 5.94 alleles per locus, with mean observed and expected heterozygosities of 0.648 and 0.620, respectively. Among of them, 15 microsatellite markers were identified without null alleles and with Hardy–Weinberg equilibrium. These alleles showed a combined non-exclusion probability of 0.01138. The probability of individual identification (P_ID) value combined with in descending order 12 microsatellite markers was 0.00008, which strongly suggests that the use of the microsatellite markers developed in this study in various combinations would result in a high resolution method for parentage analysis and individual identification. These markers could be used to establish a broodstock management program for giant grouper and to provide a foundation for genetic studies such as population structure, parentage analysis, and kinship selection.     

Effects of Peroxisome Proliferator-Activated Receptor-δ Agonist on Cardiac Healing after Myocardial Infarction

Peroxisome proliferator-activated receptor-delta (PPAR-δ)-dependent signaling is associated with rapid wound healing in the skin. Here, we investigated the therapeutic effects of PPAR-δ-agonist treatment on cardiac healing in post-myocardial infarction (MI) rats. Animals were assigned to the following groups: sham-operated control group, left anterior descending coronary artery ligation (MI) group, or MI with administration of the PPAR-δ agonist GW610742 group. GW610742 (1 mg/kg) was administrated intraperitoneally after the operation and repeated every 3 days. Echocardiographic data showed no differences between the two groups in terms of cardiac function and remodeling until 4 weeks. However, the degrees of angiogenesis and fibrosis after MI were significantly higher in the GW610742-treated rats than in the untreated MI rats at 1 week following MI, which changes were not different at 2 weeks after MI. Naturally, PPAR-δ expression in infarcted myocardium was highest increased in 3 day after MI and then disappeared in 14 day after MI. GW610742 increased myofibroblast differentiation and transforming growth factor-beta 2 expression in the infarct zone at 7 days after MI. GW610742 also increased bone marrow-derived mesenchymal stem cell (MSC) recruitment in whole myocardium, and increased serum platelet-derived growth factor B, stromal-derived factor-1 alpha, and matrix metallopeptidase 9 levels at day 3 after MI. PPAR-δ agonists treatment have the temporal effect on early fibrosis of infarcted myocardium, which might not sustain the functional and structural beneficial effect.     

Comparison of Metabolites Variation and Antiobesity Effects of Fermented versus Nonfermented Mixtures of Cudrania tricuspidata,Lonicera caerulea,and Soybean According to Fermentation In Vitro and In Vivo

We used ultra-performance-liquid-chromatography with quadrupole-time-of-flight mass spectrometry to study the changes in metabolites in the mixture of Cudrania tricuspidata, Lonicera caerulea, and soybean (CLM) during fermentation. Additionally, the antiobesity effects of CLM and fermented-CLM (FCLM) were studied based on the analysis of plasma from high-fat diet (HFD)-fed mice. The levels of cyanidin and the glycosides of luteolin, quercetin, and cyanidin derived from L. caerulea were decreased, whereas the levels of luteolin and quercetin were increased during fermentation. Isoflavone glycosides and soyasaponins originating from the soybean were decreased, whereas their aglycones such as daidzein, glycitein, and genistein were increased. As for prenylated flavonoids from C. tricuspidata, these metabolites were decreased at the early stage of fermentation, and were increased at end of the fermentation. In terms of the functional food product, various metabolites derived from diverse natural products in CLM had complementary effects and demonstrated higher antioxidant and pancreatic lipase inhibition activities after fermentation; these activities were closely related to flavonoid aglycones including genistein, daidzein, glycitein, luteolin, and quercetin. In an in vivo experiment, several clinical parameters affected by HFD were improved by the administration of either CLM or FCLM, but there was a difference in the antiobesity effects. The levels of lysoPCs with C20:4, C16:0, and C22:6 were significantly attenuated by CLM administration, while the attenuated levels of lysoPCs with C20:4 and C18:2 were significantly restored by FCLM administration. These metabolites may explain the above-mentioned differences in antiobesity effects. Although only the changes in plasma lysophospholipids could not fully explain antiobesity effects between non-fermented and fermented plant mixtures from our results, we suggest that metabolomics approach could provide a way to reveal the metabolite alterations in the complex fermentation process and understand the differences or changes in bioactivity according to fermentation.