A Model to Evaluate the Cytotoxicity of the Fungal Volatile Organic Compound 1-octen-3-ol in Human Embryonic Stem Cells

Microbial growth in damp indoor environments has been correlated with risks to human health. This study was aimed to determine the cytotoxicity of 1-octen-3-ol (“mushroom alcohol”), a major fungal volatile organic compound (VOC) associated with mushroom and mold odors. Using an airborne exposure technique, human embryonic stem cells were exposed for 1 h to different concentrations (0–1,000 ppm) of racemic 1-octen-3-ol and its enantiomers, (R)-(−)-1-octen-3-ol and (S)-(+)-1-octen-3-ol. Cytotoxicity was assayed using both the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and the fluorescently tagged Calcein AM-mediated “live and dead” assay. Racemic 1-octen-3-ol and (S)-(+)-1-octen-3-ol exhibited greater cytotoxicity to the undifferentiated human cell line H1 than did (R)-(−)-1-octen-3-ol. The inhibition concentration 50 (IC₅₀) values assessed by the MTS assay for racemic 1-octen-3-ol, (S)-(+)-1-octen-3-ol and (R)-(−)-1-octen-3-ol were, respectively, 109, 98, and 258 ppm. These IC₅₀ values were 40–80-fold lower than that of vapor phase toluene, an industrial chemical used as a positive control in this study. Our report pioneers the modeling of human embryonic stem cells as an in vitro approach to screen the potential toxicity of fungal VOCs. Human embryonic stem cells exposed to 1-octen-3-ol, and its enantiomers in the vapor phase showed more cytotoxicity than those exposed to toluene.     

Sequential evolution and escape from neutralization of simian immunodeficiency virus SIVsmE660 clones in rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques has become an important surrogate model for evaluating HIV vaccine strategies. The extreme resistance to neutralizing antibody (NAb) of many commonly used strains, such as SIVmac251/239 and SIVsmE543-3, limits their potential relevance for evaluating the role of NAb in vaccine protection. In contrast, SIVsmE660 is an uncloned virus that appears to be more sensitive to neutralizing antibody. To evaluate the role of NAb in this model, we generated full-length neutralization-sensitive molecular clones of SIVsmE660 and evaluated two of these by intravenous inoculation of rhesus macaques. All animals became infected and maintained persistent viremia that was accompanied by a decline in memory CD4(+) T cells in blood and bronchoalveolar lavage fluid. High titers of autologous NAb developed by 4 weeks postinoculation but were not associated with control of viremia, and neutralization escape variants were detected concurrently with the generation of NAb. Neutralization escape was associated with substitutions and insertion/deletion polymorphisms in the V1 and V4 domains of envelope. Analysis of representative variants revealed that escape variants also induced NAbs within a few weeks of their appearance in plasma, in a pattern that is reminiscent of the escape of human immunodeficiency virus type 1 (HIV-1) isolates in humans. Although early variants maintained a neutralization-sensitive phenotype, viruses obtained later in infection were significantly less sensitive to neutralization than the parental viruses. These results indicate that NAbs exert selective pressure that drives the evolution of the SIV envelope and that this model will be useful for evaluating the role of NAb in vaccine-mediated protection.     

Human ESC self-renewal promoting microRNAs induce epithelial-mesenchymal transition in hepatocytes by controlling the PTEN and TGFβ tumor suppressor signaling pathways

The self-renewal capacity ascribed to embryonic stem cells (ESC) is reminiscent of cancer cell proliferation, raising speculation that a common network of genes may regulate these traits. A search for general regulators of these traits yielded a set of microRNAs for which expression is highly enriched in human ESCs and liver cancer cells (HCC) but attenuated in differentiated quiescent hepatocytes. Here, we show that these microRNAs promote hESC self-renewal, as well as HCC proliferation, and when overexpressed in normally quiescent hepatocytes, induce proliferation and activate cancer signaling pathways. Proliferation in hepatocytes is mediated through translational repression of Pten, Tgfbr2, Klf11, and Cdkn1a, which collectively dysregulates the PI3K/AKT/mTOR and TGFβ tumor suppressor signaling pathways. Furthermore, aberrant expression of these miRNAs is observed in human liver tumor tissues and induces epithelial-mesenchymal transition in hepatocytes. These findings suggest that microRNAs that are essential in normal development as promoters of ESC self-renewal are frequently upregulated in human liver tumors and harbor neoplastic transformation potential when they escape silencing in quiescent human hepatocytes.     

Genome Scan of M. tuberculosis Infection and Disease in Ugandans

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is an enduring public health problem globally, particularly in sub-Saharan Africa. Several studies have suggested a role for host genetic susceptibility in increased risk for TB but results across studies have been equivocal. As part of a household contact study of Mtb infection and disease in Kampala, Uganda, we have taken a unique approach to the study of genetic susceptibility to TB, by studying three phenotypes. First, we analyzed culture confirmed TB disease compared to latent Mtb infection (LTBI) or lack of Mtb infection. Second, we analyzed resistance to Mtb infection in the face of continuous exposure, defined by a persistently negative tuberculin skin test (PTST-); this outcome was contrasted to LTBI. Third, we analyzed an intermediate phenotype, tumor necrosis factor-alpha (TNFα) expression in response to soluble Mtb ligands enriched with molecules secreted from Mtb (culture filtrate). We conducted a full microsatellite genome scan, using genotypes generated by the Center for Medical Genetics at Marshfield. Multipoint model-free linkage analysis was conducted using an extension of the Haseman-Elston regression model that includes half sibling pairs, and HIV status was included as a covariate in the model. The analysis included 803 individuals from 193 pedigrees, comprising 258 full sibling pairs and 175 half sibling pairs. Suggestive linkage (p<10⁻³) was observed on chromosomes 2q21-2q24 and 5p13-5q22 for PTST-, and on chromosome 7p22-7p21 for TB; these findings for PTST- are novel and the chromosome 7 region contains the IL6 gene. In addition, we replicated recent linkage findings on chromosome 20q13 for TB (p = 0.002). We also observed linkage at the nominal α = 0.05 threshold to a number of promising candidate genes, SLC11A1 (PTST- p = 0.02), IL-1 complex (TB p = 0.01), IL12BR2 (TNFα p = 0.006), IL12A (TB p = 0.02) and IFNGR2 (TNFα p = 0.002). These results confirm not only that genetic factors influence the interaction between humans and Mtb but more importantly that they differ according to the outcome of that interaction: exposure but no infection, infection without progression to disease, or progression of infection to disease. Many of the genetic factors for each of these stages are part of the innate immune system.     

Parameter estimation for a leaky integrate-and-fire neuronal model from ISI data

The Ornstein-Uhlenbeck process has been proposed as a model for the spontaneous activity of a neuron. In this model, the firing of the neuron corresponds to the first passage of the process to a constant boundary, or threshold. While the Laplace transform of the first-passage time distribution is available, the probability distribution function has not been obtained in any tractable form. We address the problem of estimating the parameters of the process when the only available data from a neuron are the interspike intervals, or the times between firings. In particular, we give an algorithm for computing maximum likelihood estimates and their corresponding confidence regions for the three identifiable (of the five model) parameters by numerically inverting the Laplace transform. A comparison of the two-parameter algorithm (where the time constant tau is known a priori) to the three-parameter algorithm shows that significantly more data is required in the latter case to achieve comparable parameter resolution as measured by 95% confidence intervals widths. The computational methods described here are a efficient alternative to other well known estimation techniques for leaky integrate-and-fire models. Moreover, it could serve as a template for performing parameter inference on more complex integrate-and-fire neuronal models.     

Isolation, identification and application of novel bacterial consortium TJ-1 for the decolourization of structurally different azo dyes

A novel bacterial consortium (TJ-1), which could decolorize Acid Orange 7 (AO7) and manyother azo dyes, was developed. In TJ-1 three bacterial strains were identified as Aeromonas caviae, Proteus mirabilis and Rhodococcus globerulus by 16S rRNA gene sequence analysis. AO7 decolorization was significantly higher with the use of consortium as compared to the use of individual strains, indicating complementary interactions among these strains. AO7 decolorization was observed under microaerophilic condition in the presence of organic carbon source. Either yeast extract (YE) alone or a combination of YE and glucose resulted in much higher decolorization of AO7 as compared to glucose alone, peptone or starch. Kinetic studies with different initial AO7 concentrations showed that more than 90% decolorization could be achieved even at 200mg/l within 16h. Fed-batch studies showed that AO7 decolorization required 10h during the first cycle and 5h in the second and third cycles, showing that bacterial cells could be used for multiple cycles. The consortium also decolorized fifteen other azo dyes individually as well as a simulated wastewater containing a mixture of all the sixteen azo dyes, thus, conferring the possibility of application of TJ-1 for the treatment of industrial wastewaters.     

Web Proxy Acceleration

Numerous studies show that miss ratios at forward proxies are typically at least 40–50%. This paper proposes and evaluates a new approach for improving the throughput of Web proxy systems by reducing the overhead of handling cache misses. Namely, we propose to front-end a Web proxy with a high performance node that filters the requests, processing the misses and forwarding the hits and the new cacheable content to the proxy. Requests are filtered based on hints of the proxy cache content. This system, called Proxy Accelerator, achieves significantly better communications performance than a traditional proxy system. For instance, an accelerator can be built as an embedded system optimized for communication and HTTP processing, or as a kernel-mode HTTP server. Scalability with the Web proxy cluster size is achieved by using several accelerators. We use analytical models, trace-based simulations, and a real implementation to study the benefits and the implementation tradeoffs of this new approach. Our results show that a single proxy accelerator node in front of a 4-node Web proxy can improve the cost-performance ratio by about 40%. Hint-based request filter implementation choices that do not affect the overall hit ratio are available. An implementation of the hint management module integrated in Web proxy software is presented. Experimental evaluation of the implementation demonstrates that the associated overheads are very small.     

Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin

Background

Although genomic DNA isolation using the Chelex 100 resin is rapid and inexpensive, the DNA obtained by this method has a low concentration in solution and contains suspended impurities. The presence of debris in the DNA solution may result in degradation of DNA on long term storage and inhibition of the polymerase chain reaction. In order to remove impurities and concentrate the DNA in solution, we have introduced modifications in the existing DNA isolation protocol using Chelex-100. We used ammonium acetate to precipitate proteins and a sodium acetate- isopropanol mixture to pellet out DNA which was washed with ethanol.

Results

A pure DNA pellet that can be dissolved in water or Tris-EDTA buffer and stored for a long time at ??80 °C was obtained. We also observed a 20-fold change in the DNA concentration following precipitation and re-dissolution.

Conclusion

Our method is different from other extraction methods since it uses non-toxic, easily available and inexpensive reagents as well as minimal amounts of blood or tissue samples for the DNA extraction process. Besides its use in sex determination and genotyping in lab animals as described in this paper, it may also have applications in forensic science and diagnostics such as the easy detection of pathogenic DNA in blood.