High throughput screening of hydrolytic enzymes from termites using a natural substrate derived from sugarcane bagasse

Background

Short rotation coppice willow is a potential lignocellulosic feedstock in the United Kingdom and elsewhere; however, research on optimising willow specifically for bioethanol production has started developing only recently. We have used the feedstock Salix viminalis × Salix schwerinii cultivar 'Olof' in a three-month pot experiment with the aim of modifying cell wall composition and structure within the stem to the benefit of bioethanol production. Trees were treated for 26 or 43 days with tension wood induction and/or with an application of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile that is specific to secondary cell walls. Reaction wood (tension and opposite wood) was isolated from material that had received the 43-day tension wood induction treatment.

Results

Glucan content, lignin content and enzymatically released glucose were assayed. All measured parameters were altered without loss of total stem biomass yield, indicating that enzymatic saccharification yield can be enhanced by both alterations to cell wall structure and alterations to absolute contents of either glucan or lignin.

Conclusions

Final glucose yields can be improved by the induction of tension wood without a detrimental impact on biomass yield. The increase in glucan accessibility to cell wall degrading enzymes could help contribute to reducing the energy and environmental impacts of the lignocellulosic bioethanol production process.     

Hematopoietic stem cell gene therapy: dead or alive?

Despite some reports of toxicity in recent clinical trials, many scientists believe that the use of gene therapy in the treatment of congenital genetic defects and acquired disorders has too much potential to abandon. Hematopoietic stem cells (HSCs) have been primary targets for gene therapy owing to their capacity for differentiation and self-renewal, whereby multiple cell lineages can potentially be corrected for the lifetime of an individual. These efforts represent a long-term investment towards broadening physicians' treatment options for patients whose diseases, in particular certain immunodeficiencies, are fatal and where no other therapy is available. We review the recent progress and clinical triumphs as well as the reported toxicity related to insertional mutagenesis. We also discuss the current risk-to-benefit estimates and future strategies to reduce the risks and allow full realization of clinical potential. Scientists are continually revising protocols: going both from "bench to bedside" and, as strikingly demonstrated by HSC gene therapy, from "bedside to bench."     

Genome-wide expression analysis of iron regulation in Burkholderia pseudomallei and Burkholderia mallei using DNA microarrays

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. As iron regulation of gene expression is common in bacteria, in the present studies, we have used microarray analysis to examine the effects of growth in different iron concentrations on the regulation of gene expression in B. pseudomallei and B. mallei. Gene expression profiles for these two bacterial species were similar under high and low iron growth conditions irrespective of growth phase. Growth in low iron led to reduced expression of genes encoding most respiratory metabolic systems and proteins of putative function, such as NADH-dehydrogenases, cytochrome oxidases, and ATP-synthases. In contrast, genes encoding siderophore-mediated iron transport, heme-hemin receptors, and a variety of metabolic enzymes for alternative metabolism were induced under low iron conditions. The overall gene expression profiles suggest that B. pseudomallei and B. mallei are able to adapt to the iron-restricted conditions in the host environment by up-regulating an iron-acquisition system and by using alternative metabolic pathways for energy production. The observations relative to the induction of specific metabolic enzymes during bacterial growth under low iron conditions warrants further experimentation.     

Waist-to-hip ratio versus body mass index as predictors of fitness in women

The claim that men prefer women with low waist-to-hip ratios (WHR) has been vigorously disputed. We examine self-report data from 359 primiparous Polish women (with normal singleton births and healthy infants) and show that WHR correlates with at least one component of a woman’s biological fitness (her first child’s birth weight, a variable that significantly affects infant survival rates). However, a woman’s Body Mass Index (BMI) is a better predictor of her child’s neonatal weight in small-bodied women (<54 kg). The failure to find a preference for low WHR in some traditional populations may thus be a consequence of the fact that, even in western populations, body mass is a better predictor of fitness in those cases characterized by low maternal body weight. Boguslaw Pawłowski Ph.D., D.Sc., is a researcher and lecturer in biological anthropology at the University of Wrocław, Poland. His research interests focus on mechanisms of human evolution (with special attention to the evolution of subcutaneous fat tissue distribution) and human mate choice. Robin Dunbar Ph.D., FBA is British Academy Research Professor of Evolutionary Psychology at the University of Liverpool, England, and co-Director of the British Academy Centenary Research Project. His research interests focus on the evolutionary and environmental determinants of social and reproductive strategies, with particular references to humans, nonhuman primates, and ungulates.     

ADAM10 mediates ectodomain shedding of the betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.     

2.45 GHz radiofrequency fields alter gene expression in cultured human cells

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2-h exposure. The number of affected genes increased to 759 after a 6-h exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.     

Immunodominance of poxviral-specific CTL in a human trial of recombinant-modified vaccinia Ankara

Many recombinant poxviral vaccines are currently in clinical trials for cancer and infectious diseases. However, these agents have failed to generate T cell responses specific for recombinant gene products at levels comparable with T cell responses associated with natural viral infections. The recent identification of vaccinia-encoded CTL epitopes, including a new epitope described in this study, allows the simultaneous comparison of CTL responses specific for poxviral and recombinant epitopes. We performed detailed kinetic analyses of CTL responses in HLA-A*0201 patients receiving repeated injections of recombinant modified vaccinia Ankara encoding a string of melanoma tumor Ag epitopes. The vaccine-driven CTL hierarchy was dominated by modified vaccinia Ankara epitope-specific responses, even in patients who had not received previous smallpox vaccination. The only recombinant epitope that was able to impact on the CTL hierarchy was the melan-A26-35 analog epitope, whereas responses specific for the weaker affinity epitope NY-ESO-1(157-165) failed to be expanded above the level detected in prevaccination samples. Our results demonstrate that immunodominant vaccinia-specific CTL responses limit the effectiveness of poxviruses in recombinant vaccination strategies and that more powerful priming strategies are required to overcome immunodominance of poxvirus-specific T cell responses.     

Designs for single- or multiple-agent phase I trials

Phase I trials of cytotoxic agents in oncology are usually dose-finding studies that involve a single cytotoxic agent. Many statistical methods have been proposed for these trials, all of which are based on the assumption of a monotonic dose-toxicity curve. For single-agent trials, this is a valid assumption. In many trials, however, investigators are interested in finding the maximally tolerated dose based on escalating multiple cytotoxic agents. When there are multiple agents, monotonicity of the dose-toxicity curve is not clearly defined. In this article we present a design for phase I trials in which the toxicity probabilities follow a partial order, meaning that there are pairs of treatments for which the ordering of the toxicity probabilities is not known at the start of the trial. We compare the new design to existing methods for simple orders and investigate the properties of the design for two partial orders.