Downregulation of 14-3-3 Proteins in a Kainic Acid-Induced Neurotoxicity Model

The 14-3-3 proteins are among the most abundant proteins expressed in the brain, comprising about 1% of the total amount of soluble brain proteins. Through phosphoserine- and phosphothreonine-binding motifs, 14-3-3 proteins regulate many signaling proteins and cellular processes including cell death. In the present study, we utilized a well-known kainic acid (KA)-induced excitotoxicity rat model and examined the expression of 14-3-3 and its isoforms in the frontal cortex of KA-treated and control animals. Among the different 14-3-3 isoforms, abundant levels of eta and tau were detected in the frontal cortex, followed by sigma, epsilon, and gamma, while the expression levels of alpha/beta and zeta/delta isoforms were low. Compared to the control animals, KA treatment induced a significant downregulation of the overall 14-3-3 protein level as well as the levels of the abundant isoforms eta, tau, epsilon, and gamma. We also investigated two 14-3-3-interacting proteins that are involved in the cell death process: Bcl-2-associated X (BAX) and extracellular signal-regulated kinase (ERK). Both BAX and phosphorylated ERK showed increased levels following KA treatment. Together, these findings demonstrate an abundance of several 14-3-3 isoforms in the frontal cortex and that KA treatment can cause a downregulation of 14-3-3 expression and an upregulation of 14-3-3-interacting proteins BAX and phospho-ERK. Thus, downregulation of 14-3-3 proteins could be one of the early molecular events associated with excitotoxicity. This could lead to subsequent upregulation of 14-3-3-binding proteins such as BAX and phospho-ERK that contribute to further downstream apoptosis processes, eventually leading to cell death. Maintaining sufficient levels of 14-3-3 expression and function may become a target of therapeutic intervention for excitotoxicity-induced neurodegeneration.     

Decreasing mortality and hospitalizations with rising costs related to gastric cancer in the USA: an epidemiological perspective

Background

There is no convincing data on the trends of hospitalizations, mortality, cost, and demographic variations associated with inpatient admissions for gastric cancer in the USA. The aim of this study was to use a national database of US hospitals to evaluate the trends associated with gastric cancer.

Methods

We analyzed the National Inpatient Sample (NIS) database for all patients in whom gastric cancer (ICD-9 code: 151.0, 151.1, 151.2, 151.3, 151.4, 151.5, 151.6, 151.8, 151.9) was the principal discharge diagnosis during the period, 2003–2014. The NIS is the largest publicly available all-payer inpatient care database in the US. It contains data from approximately eight million hospital stays each year. The statistical significance of the difference in the number of hospital discharges, length of stay, and hospital costs over the study period was determined by regression analysis.

Results

In 2003, there were 23,921 admissions with a principal discharge diagnosis of gastric cancer as compared to 21,540 in 2014 (P?<?0.01). The mean length of stay for gastric cancer decreased by 17% between 2003 and 2014 from 10.9?days to 8.95?days (P?<?0.01). However, during this period, the mean hospital charges increased significantly by 21% from $ 75,341 per patient in 2003 to $ 91,385 per patient in 2014 (P?<?0.001). There was a more significant reduction in mortality over a period of 11?years from 2428 (10.15%) in 2003 to 1345 (6.24%) in 2014 (P?<?0.01). The aggregate charges (i.e., “national bill”) for gastric cancer increased significantly from 1.79 bn $ to 1. 96 bn $ (P?<?0.001), despite decrease in hospitalization (inflation adjusted).

Conclusion

Although the number of inpatient admissions for gastric cancer have decreased over the past decade, the healthcare burden and cost related to it has increased significantly. Inpatient mortality is decreasing which is consistent with overall decrease in gastric cancer-related deaths. Cost increase associated with gastric cancer contributed significantly to the national healthcare bill.

     

Development of a RapidFire mass spectrometry assay and a fluorescence assay for the discovery of kynurenine aminotransferase II inhibitors to treat central nervous system disorders

Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in the tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and, thus, is an attractive target to treat CNS diseases. A sensitive, high-throughput, label-free RapidFire mass spectrometry assay has been developed for human KATII. Unlike other assays, this method is directly applicable to KATII enzymes from different animal species, which allows us to select proper animal model(s) to evaluate human KATII inhibitors. We also established a coupled fluorescence assay for human KATII. The short assay time and kinetic capability of the fluorescence assay provide a useful tool for orthogonal inhibitor validation and mechanistic studies.     

Conservation of the C-type lectin fold for accommodating massive sequence variation in archaeal diversity-generating retroelements

Background

Diversity-generating retroelements (DGRs) provide organisms with a unique means for adaptation to a dynamic environment through massive protein sequence variation. The potential scope of this variation exceeds that of the vertebrate adaptive immune system. DGRs were known to exist only in viruses and bacteria until their recent discovery in archaea belonging to the ‘microbial dark matter’, specifically in organisms closely related to Nanoarchaeota. However, Nanoarchaeota DGR variable proteins were unassignable to known protein folds and apparently unrelated to characterized DGR variable proteins.

Results

To address the issue of how Nanoarchaeota DGR variable proteins accommodate massive sequence variation, we determined the 2.52 Å resolution limit crystal structure of one such protein, AvpA, which revealed a C-type lectin (CLec)-fold that organizes a putative ligand-binding site that is capable of accommodating 10¹³ sequences. This fold is surprisingly reminiscent of the CLec-folds of viral and bacterial DGR variable protein, but differs sufficiently to define a new CLec-fold subclass, which is consistent with early divergence between bacterial and archaeal DGRs. The structure also enabled identification of a group of AvpA-like proteins in multiple putative DGRs from uncultivated archaea. These variable proteins may aid Nanoarchaeota and these uncultivated archaea in symbiotic relationships.

Conclusions

Our results have uncovered the widespread conservation of the CLec-fold in viruses, bacteria, and archaea for accommodating massive sequence variation. In addition, to our knowledge, this is the first report of an archaeal CLec-fold protein.

     

Photoluminescence Study of Silver Nano-hexagons

Plasmonics - Silver nano-hexagons have been prepared through the reduction of silver ions by ethylene glycol with poly (vinyl pyrrolidone) (PVP) as a capping agent or stabilizing agent. The...     

Trends in the Care of Diabetic Macular Edema: Analysis of a National Cohort

Purpose

To evaluate how the monitoring and treatment for diabetic macular edema (DME) has changed in a national sample.

Design

Retrospective cohort study.

Methods

Setting: Administrative medical claims data from a large, national U.S. insurer. Study population: Beneficiaries of a U.S. insurance company. Observation procedures: All incident cases of DME were found. Those in years 2002/3, 2006 and 2010 were followed for a 2-year observation period and those from 2009, 2010 and 2011 for a 1-year observation period. Main Outcome Measures: Types and frequencies of treatment were tallied and compared over each of the cohorts.

Results

Two-year cohorts had 233, 251 and 756 patients in 2002/3, 2006 and 2010 respectively. One-year cohorts had 1002, 1119 and 1382 patients in 2009, 2010 and 2011, respectively. Both percentage of patients receiving therapy and number of treatments given increased across the 2-year cohorts for both focal laser and anti-vascular endothelial growth factor (anti-VEGF) (p<0.001). The highest use of anti-VEGF agents in any of the cohorts was in the 2011 1-year group that only averaged 3.78 injections. Focal laser was used 2.5x as frequently as anti-VEGF injections in the most recent cohorts with only a high of 14.0% of DME patients receiving anti-VEGF therapy in any of the cohorts.

Conclusion

Regardless of treatment modality (laser or injection) DME patients received vastly fewer treatments than patients in randomized control trials. Despite the proven superior visual outcomes of anti-VEGF agents over focal laser in DME, focal laser was still used more frequently.     

Race/Ethnicity-Specific Association of Vitamin D and Global DNA Methylation: Cross-Sectional and Interventional Findings

ObjectivesUnderstanding of the influence of vitamin D deficiency on epigenome will provide novel insights into the chronic disease risk. We tested our hypotheses that 1) vitamin D deficiency is associated with global hypomethylation and this association may be race/ethnicity dependent; and 2) vitamin D supplementation will increase global DNA methylation level.MethodsA two-stage design, cross-sectional observation followed by a 16 week randomized, double- blinded, placebo-controlled trial (RCT) of vitamin D3 supplementation, was undertaken. Global DNA methylation level (percentage of 5-methylcytosine, %5-mC) was quantified using leukocyte DNA with the MethylFlash^TM Methylated DNA Quantification kit (Epigentek). Global methylation data was obtained from 454 Caucasians and African Americans (42%) in the observation cohort and 58 African Americans with vitamin D deficiency in the dose responsive RCT.ResultsIn the cross-sectional study, African Americans had lower %5-mC than Caucasians (P = 0.04). A significant interaction was detected between plasma 25(OH)D and race on %5-mC (P = 0.05), as a positive association was observed between plasma 25(OH)D and %5-mC in African Americans (β = 0.20, p<0.01), but not in Caucasians (β = 0.03, p = 0.62). In the 16-week RCT, a dose-response benefit of vitamin D₃ supplementation was observed for %5-mC, as indicated by a significant linear upward trend (-0.01 ± 0.01%, placebo; 0.11 ± 0.01%, ~600 IU/day; 0.30 ± 0.01%, ~2,000 IU/day; and 0.65 ± 0.01%, ~4,000 IU/day group; P-trend = 0.04).ConclusionsVitamin D deficiency is associated with global hypomethylation in African Americans. Vitamin D3 supplementation increases global DNA methylation in a dose-response manner in African Americans with vitamin D deficiency.     

De Novo Occurrence of a Variant in ARL3 and Apparent Autosomal Dominant Transmission of Retinitis Pigmentosa

Background

Retinitis pigmentosa is a phenotype with diverse genetic causes. Due to this genetic heterogeneity, genome-wide identification and analysis of protein-altering DNA variants by exome sequencing is a powerful tool for novel variant and disease gene discovery. In this study, exome sequencing analysis was used to search for potentially causal DNA variants in a two-generation pedigree with apparent dominant retinitis pigmentosa.

Methods

Variant identification and analysis of three affected members (mother and two affected offspring) was performed via exome sequencing. Parental samples of the index case were used to establish inheritance. Follow-up testing of 94 additional retinitis pigmentosa pedigrees was performed via retrospective analysis or Sanger sequencing.

Results and Conclusions

A total of 136 high quality coding variants in 123 genes were identified which are consistent with autosomal dominant disease. Of these, one of the strongest genetic and functional candidates is a c.269A>G (p.Tyr90Cys) variant in ARL3. Follow-up testing established that this variant occurred de novo in the index case. No additional putative causal variants in ARL3 were identified in the follow-up cohort, suggesting that if ARL3 variants can cause adRP it is an extremely rare phenomenon.     

Relative hepatotoxicity of ortho and meta mono substituted thiobenzamides in the rat

Thiobenzamide is known to be hepatotoxic in the rat and the relative hepatotoxicity of para-substituted thiobenzamides has previously been shown to depend strictly on the electronic character of the para substituent. We have now extended this study to include ortho and meta monosubstituted thiobenzamides. Among the meta-substituted compounds, hepatotoxicity varies in strict accordance with the electronic character of the substituent, whereas the ortho-substituted compounds show no toxicity at comparable doses regardless of the nature of the substituent. Explanations for these substituent effects are provided in terms of the chemical reactivity of the compounds and their corresponding S-oxide and S,S-dioxide metabolites.     

The addition of hypobromous acid to 3β,17β-diacetoxy-4-estrene and an equilibration study involving neighboring group participation by the a-ring acetoxy group

The reaction of 3β,17β-diacetoxy-4-estrene with N-bromoacetamide in a two phase ether/water solvent mixture gave 5-bromo-4β,17β-diacetoxy-5α-estran-3β-ol as the major product (75%). Four minor products were also isolated and identified. These were: 4α-bromo-3β, 17β-diacetoxy-5α-estran-5-ol (5%), 5-bromo-3g,17β-diacetoxy-5α-estran-4β-ol (6%), 5-bromo-4α,17β-diacetoxy-5αt-estran-3β-ol (3%), and 4β-bromo-3β,17β-diacetoxy-5α-estran-5-ol (4%). The 5-bromo-4β, 17β-diacetoxy-5α-estran-3β-ol was equilibrated by heating with oxalic acid in refluxing benzene for $\text{ca}$. 16h to give a mixture of it and 5-broino-3β,17β-diacetoxy-5α-estran-4β-ol in the ratio of 16:84 respectively. A similar equilibration mixture (14:86) was obtained under identical conditions when 5-bromo-3β,17β-diacetoxy-5α-estran-4β-ol was the starting material.