Embryonic requirements for ErbB signaling in neural crest development and adult pigment pattern formation

Vertebrate pigment cells are derived from neural crest cells and are a useful system for studying neural crest-derived traits during post-embryonic development. In zebrafish, neural crest-derived melanophores differentiate during embryogenesis to produce stripes in the early larva. Dramatic changes to the pigment pattern occur subsequently during the larva-to-adult transformation, or metamorphosis. At this time, embryonic melanophores are replaced by newly differentiating metamorphic melanophores that form the adult stripes. Mutants with normal embryonic/early larval pigment patterns but defective adult patterns identify factors required uniquely to establish, maintain or recruit the latent precursors to metamorphic melanophores. We show that one such mutant, picasso, lacks most metamorphic melanophores and results from mutations in the ErbB gene erbb3b, which encodes an EGFR-like receptor tyrosine kinase. To identify critical periods for ErbB activities, we treated fish with pharmacological ErbB inhibitors and also knocked down erbb3b by morpholino injection. These analyses reveal an embryonic critical period for ErbB signaling in promoting later pigment pattern metamorphosis, despite the normal patterning of embryonic/early larval melanophores. We further demonstrate a peak requirement during neural crest migration that correlates with early defects in neural crest pathfinding and peripheral ganglion formation. Finally, we show that erbb3b activities are both autonomous and non-autonomous to the metamorphic melanophore lineage. These data identify a very early, embryonic, requirement for erbb3b in the development of much later metamorphic melanophores, and suggest complex modes by which ErbB signals promote adult pigment pattern development.     

False discovery rate, sensitivity and sample size for microarray studies

MOTIVATION: In microarray data studies most researchers are keenly aware of the potentially high rate of false positives and the need to control it. One key statistical shift is the move away from the well-known P-value to false discovery rate (FDR). Less discussion perhaps has been spent on the sensitivity or the associated false negative rate (FNR). The purpose of this paper is to explain in simple ways why the shift from P-value to FDR for statistical assessment of microarray data is necessary, to elucidate the determining factors of FDR and, for a two-sample comparative study, to discuss its control via sample size at the design stage. RESULTS: We use a mixture model, involving differentially expressed (DE) and non-DE genes, that captures the most common problem of finding DE genes. Factors determining FDR are (1) the proportion of truly differentially expressed genes, (2) the distribution of the true differences, (3) measurement variability and (4) sample size. Many current small microarray studies are plagued with large FDR, but controlling FDR alone can lead to unacceptably large FNR. In evaluating a design of a microarray study, sensitivity or FNR curves should be computed routinely together with FDR curves. Under certain assumptions, the FDR and FNR curves coincide, thus simplifying the choice of sample size for controlling the FDR and FNR jointly.     

Prophylactic alpha interferon treatment increases the therapeutic index of oncolytic vesicular stomatitis virus virotherapy for advanced hepatocellular carcinoma in immune-competent rats

Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with intrinsic oncolytic specificity due to substantially attenuated antiviral responses in many tumors. We have recently reported that recombinant VSV vector can be used as an effective oncolytic agent to safely treat multifocal hepatocellular carcinoma (HCC) in the livers of immune-competent rats via hepatic artery infusion. When administered at doses above the maximum tolerated dose (MTD), however, the animals suffered from neurotoxicity and/or acute lethal hepatotoxicity. Since VSV is extremely sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta) in normal cells, we tested if prophylactic treatment with rat IFN-alpha would enhance VSV safety without compromising treatment efficacy in tumor-bearing rats. We found that VSV retained its replication potential in human and rat HCC cells after preincubation with relatively high doses of rat and human IFN-alpha in vitro, and its MTD in tumor-bearing rats treated systemically with rat IFN-alpha at 66 IU/g body weight (BW), equivalent to a human IFN-alpha dose that is currently prescribed for patients with viral hepatitis, was elevated by more than 1/2 log unit. Furthermore, we demonstrate that intratumoral replication of VSV was not attenuated by administration of 66 IU/g BW rat IFN-alpha, as tumor response and survival advantage in VSV-treated rats in the presence or absence of rat IFN-alpha were equivalent. The results suggest that prophylactic rat IFN-alpha treatment elevates the therapeutic index of hepatic arterial VSV therapy for multifocal HCC in rats. Since human IFN-alpha is currently in clinical use, its prophylactic application should be considered in future clinical translational protocols for VSV-mediated oncolytic virotherapy as a novel therapeutic modality in patients with advanced HCC, as well as other types of cancer.     

Role of histone deacetylation in cell-specific expression of endothelial nitric-oxide synthase

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of endothelial nitric-oxide synthase (eNOS) are not fully understood. In this study we investigated whether histone deacetylation was involved in repression of eNOS expression in non-endothelial cells. Induction of eNOS expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate was observed in all four different types of non-endothelial cells examined. Chromatin immunoprecipitation assays showed that the induction of eNOS expression by TSA was accompanied by a remarkable increase of acetylation of histone H3 associated with the eNOS 5'-flanking region in the non-endothelial cells. Moreover, DNA methylation-mediated repression of eNOS promoter activity was partially reversed by TSA treatment, and combined treatment of TSA and 5-aza-2'-deoxycytidine (AzadC) synergistically induced eNOS expression in non-endothelial cells. The proximal Sp1 site is critical for basal activity of eNOS promoter. The induction of eNOS by inhibition of HDACs in non-endothelial cells, however, appeared not mediated by the changes in Sp1 DNA binding activity. We further showed that Sp1 bound to the endogenous eNOS promoter and associated with HDAC1 in non-endothelial HeLa cells. Combined TSA and AzadC treatment increased Sp1 binding to the endogenous eNOS promoter but decreased the association between HDAC1 and Sp1 in HeLa cells. Our data suggest that HDAC1 plays a critical role in eNOS repression, and the proximal Sp1 site may serve a key target for HDCA1-mediated eNOS repression in non-endothelial cells.     

Engineering a chimeric pyrroloquinoline quinone glucose dehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity

An engineered Escherichia coli PQQ glucose dehydrogenase (PQQGDH) with improved enzymatic characteristics was constructed by substituting and combining the gene-encoding protein regions responsible for EDTA tolerance, thermal stability and substrate specificity. The protein region responsible for complete EDTA tolerance in Acinetobacter calcoaceticus, which is recognized as the indicator of high stability in co-factor binding, was elucidated. The region is located between 32 and 59% from the N-terminus of A. calcoaceticus PQQGDH(A27 region) and also corresponds to the same position from 32 to 59% from the N-terminus in E. coli PQQGDH, though E. coli PQQGDH is EDTA sensitive. We previously reported that the C-terminal 3% region of A. calcoaceticus (A3 region) played an important role in the increase of thermal stability, and that His775Asn substitution in E. coli PQQGDH resulted in an increase in the substrate specificity of E. coli PQQGDH towards glucose. Based on these findings, chimeric and/or mutated PQQGDHs, E97A3 H775N, E32A27E41 H782N, E32A27E38A3 and E32A27E38A3 H782N were constructed to investigate the compatibility of two protein regions and one amino acid substitution. His775 substitution to Asn corresponded to His782 substitution to Asn (H782N) in chimeric enzymes harbouring the A27 region. Since all the chimeric PQQGDHs harbouring the A27 region were EDTA tolerant, the A27 region was found to be compatible with the other region and substituted amino acid responsible for the improvement of enzymatic properties. The contribution of the A3 region to thermal stability complemented the decrease in the thermal stability due to the His775 or His782 substitution to Asn. E32A27E38A3 H782N, which harbours all the above mentioned three regions, showed improved EDTA tolerance, thermal stability and substrate specificity. These results suggested a strategy for the construction of a semi-artificial enzyme by substituting and combining the gene-encoding protein regions responsible for the improvement of enzyme characteristics. The characteristics of constructed chimeric PQQGDH are discussed based on the predicted model, beta-propeller structure.     

Life cycle assessment research and application in Indonesia

The International Journal of Life Cycle Assessment - This paper presents a review of the research and application of life cycle assessment (LCA) in Indonesia over the last 20 years and...     

Identification of differentially expressed genes and false discovery rate in microarray studies

PURPOSE OF REVIEW: To highlight the development in microarray data analysis for the identification of differentially expressed genes, particularly via control of false discovery rate. RECENT FINDINGS: The emergence of high-throughput technology such as microarrays raises two fundamental statistical issues: multiplicity and sensitivity. We focus on the biological problem of identifying differentially expressed genes. First, multiplicity arises due to testing tens of thousands of hypotheses, rendering the standard P value meaningless. Second, known optimal single-test procedures such as the t-test perform poorly in the context of highly multiple tests. The standard approach of dealing with multiplicity is too conservative in the microarray context. The false discovery rate concept is fast becoming the key statistical assessment tool replacing the P value. We review the false discovery rate approach and argue that it is more sensible for microarray data. We also discuss some methods to take into account additional information from the microarrays to improve the false discovery rate. SUMMARY: There is growing consensus on how to analyse microarray data using the false discovery rate framework in place of the classical P value. Further research is needed on the preprocessing of the raw data, such as the normalization step and filtering, and on finding the most sensitive test procedure.     

Determination of serum aflatoxin B<Subscript>1</Subscript>-lysine to evaluate the efficacy of an aflatoxin-adsorbing feed additive in pigs fed an aflatoxin B<Subscript>1</Subscript>-contaminated diet

In this study, serum aflatoxin B₁ (AFB₁)-lysine was determined in order to evaluate the in vivo efficacy of a hydrated sodium calcium aluminosilicate (HSCAS) in pigs fed AFB₁. Twenty-four 49-day-old crossbred barrows were maintained in individual cages and allowed ad libitum access to feed and water. A completely randomized design was used with six animals assigned to each of four dietary treatments for 21 days as follows: (A) basal diet (BD), (B) BD supplemented with 0.5 % HSCAS, (C) BD supplemented with 1.1 mg/kg AFB₁, and (D) BD supplemented with 0.5 % HSCAS and 1.1 mg/kg AFB₁. HSCAS was able to alleviate the toxic effects of AFB₁ on pigs and reduce (P < 0.05) the levels of serum AFB₁-lysine. Cumulative reductions of adduct yield values, calculated through the equation [(pg AFB₁-lysine/mg albumin) / (μg AFB₁/kg body weight)], were 53.0, 62.8, and 72.1 after 7, 14, and 21 days of oral exposure, respectively. AFB₁-lysine has potential as an AFB₁-specific biomarker for diagnostic purposes and for evaluating the efficacy of chemoprotective interventions in pigs.     

Predictive value of sperm deoxyribonucleic acid (DNA) fragmentation index in male infertility

Background

Recently, damage to the sperm DNA has been studied as it is associated with reduced fertilization rates, embryo quality, and pregnancy rates, also higher rates of spontaneous miscarriage.

Objective

To develop a diagnostic method in predicting male infertility.

Material and Methods

The design of this study is cross-sectional. Data were retrieved from medical records of Yasmin IVF Clinic Dr. Cipto Mangunkusumo General Hospital and Daya Medika Infertility Clinic from January to December 2015. Subjects were selected by consecutive sampling and divided into two groups: infertile and fertile. Sperm deoxyribonucleic acid fragmentation index (DFI) was determined by sperm chromatin dispersion (SCD) method using Halosperm® Kit.

Results

There were 114 subjects (36 fertile and 78 infertile) selected into this study. We found no significant difference in the age between both of groups. The median value of sperm DFI in infertile group was significantly higher, 29.95 (26.6–34.3)%, compared to 19.90 (15.6–24.4)% of the fertile group, with p?<?0.001. Area Under Curve (AUC) of sperm DFI, 0.862 (95% CI 0.783, 0.941), was higher than concentration (AUC 0.744; 95% CI 0.657, 0.831), motility (AUC 0.668; 95% CI 0.572, 0.765), and morphology (AUC 0.718; 95% CI 0.697, 0.864) of the semen analysis. At the cut-off point of 26.1%, the sperm DFI had sensitivity of 80.8% (95% CI; 70.0, 88.5), specificity of 86.1% (95% CI; 69.7, 94.8), positive predictive value (PPV) of 92.6% (95% CI; 83.0, 97.3), negative predictive value (NPV) of 67.4% (95% CI; 51.9, 80.0), positive likelihood ratio (PLR) of 12.6 (95% CI; 5.4, 29.4), and negative likelihood ratio (NLR) of 0.48 (95% CI 0.31, 0.75). Sperm DFI of ≥26.1% had prevalence ratio of 2.84 (95% CI 1.86, 4.33) for the occurrence of male infertility.

Conclusion

There was significant difference between the median value of sperm DFI of infertile men and fertile men. Compared to semen analysis, sperm DFI at cut-off point of 26.1% has a higher diagnostic value (AUC).