How to establish a lasting relationship with your host: lessons learned from Mycobacterium spp

Mycobacterium spp. enjoy an intracellular lifestyle that is fatal to most microorganisms. Bacilli persist and multiply within mononuclear phagocytes in the face of defences ranging from toxic oxygen and nitrogen radicals, acidic proteases and bactericidal peptides. Uptake of Mycobacterium by phagocytes results in the de novo formation of a phagosome, which is manipulated by the pathogen to accommodate its needs for intracellular survival and replication. The present review describes the intracellular compartment occupied by Mycobacterium spp. and presents current ideas on how mycobacteria may establish this niche, placing special emphasis on the involvement of mycobacterial cell wall lipids.     

Lessons learned from the transformation of natural product discovery to a genome-driven endeavor

Natural product discovery is currently undergoing a transformation from a phenotype-driven field to a genotype-driven one. The increasing availability of genome sequences, coupled with improved techniques for identifying biosynthetic gene clusters, has revealed that secondary metabolomes are strikingly vaster than previously thought. New approaches to correlate biosynthetic gene clusters with the compounds they produce have facilitated the production and isolation of a rapidly growing collection of what we refer to as “reverse-discovered” natural products, in analogy to reverse genetics. In this review, we present an extensive list of reverse-discovered natural products and discuss seven important lessons for natural product discovery by genome-guided methods: structure prediction, accurate annotation, continued study of model organisms, avoiding genome-size bias, genetic manipulation, heterologous expression, and potential engineering of natural product analogs.     

Distinct RNA structural domains cooperate to maintain a specific cleavage site in the 3'-UTR of IGF-II mRNAs

The insulin-like growth factor II mRNAs are targets for site-specific endonucleolytic cleavage in the 3'-UTR, which results in a very stable 3' cleavage product of 1.8 kb, consisting of 3'-UTR sequences and a poly(A) tail. The 5' cleavage product contains the coding region and is rapidly degraded. Thus, cleavage is thought to provide an additional way to control IGF-II protein synthesis. We had established that cleavage requires two widely separated sequence elements (I and II) in the 3'-UTR that form a stable duplex of 83 nucleotides. The cleavage-site itself is located in an internal loop preceded by two stable stem-loop structures. Furthermore, in a study which was based on RNA folding algorithms, we have shown that there are specific sequence and structural requirements for the cleavage reaction. Here, the functions of the different structural domains in cleavage were assessed by deletion/mutational analyses, and biochemical structure probing assays were performed to characterize better the RNA structures formed and to verify the computer folding predictions. The data suggest that the stem-loop domain contributes to maintain a highly specific c leavage-site by preventing the formation of alternative structures in the cleavage-site domain. Involvement of the nucleotides in the cleavage-site loop itself in non-Watson-Crick interactions may be important for providing a specific recognition surface for an endoribonuclease activity.     

Lessons to be learned from primary renal cell carcinomas: novel tumor antigens and HLA ligands for immunotherapy

The lack of sufficient well-defined tumor-associated antigens is still a drawback on the way to a cytotoxic T-lymphocyte-based immunotherapy of renal cell carcinoma (RCC). We are trying to define a larger number of such targets by a combined approach involving HLA ligand characterization by mass spectrometry and gene expression profiling by oligonucleotide microarrays. Here, we present the results of a large-scale analysis of 13 RCC specimens. We were able to identify more than 700 peptides, mostly from self-proteins without any evident tumor association. However, some HLA ligands derived from previously known tumor antigens in RCC. In addition, gene expression profiling of tumors and a set of healthy tissues revealed novel candidate RCC-associated antigens. For several of them, we were able to characterize HLA ligands after extraction from the tumor tissue. Apart from universal RCC antigens, some proteins seem to be appropriate candidates in individual patients only. This underlines the advantage of a personalized therapeutic approach. Further analyses will contribute additional HLA ligands to this repertoire of universal as well as patient-individual tumor antigens.Tobias Krüger and Oliver Schoor contributed equally to this work.     

Lessons learned from the introduction of genetically engineered crops: relevance to gene drive deployment in Africa

The application of gene drives to achieve public health goals, such as the suppression of Anopheles gambiae populations, or altering their ability to sustain Plasmodium spp. infections, has received much attention from researchers. If successful, this genetic tool can contribute greatly to the wellbeing of people in regions severely affected by malaria. However, engineered gene drives are a product of genetic engineering, and the experience to date, gained through the deployment of genetically engineered (GE) crops, is that GE technology has had difficulty receiving public acceptance in Africa, a key region for the deployment of gene drives. The history of GE crop deployment in this region provides good lessons for the deployment of gene drives as well. GE crops have been in commercial production for 24 years, since the planting of the first GE soybean crop in 1996. During this time, regulatory approvals and farmer adoption of these crops has grown rapidly in the Americas, and to a lesser extent in Asia. Their safety has been recognized by numerous scientific organizations. Economic and health benefits have been well documented in the countries that have grown them. However, only one transgenic crop event is being grown in Europe, and only in two countries in that region. Europe has been extremely opposed to GE crops, due in large part to the public view of agriculture that opposes “industrial” farming. This attitude is reflected in a highly precautionary regulatory and policy environment, which has highly influenced how African countries have dealt with GE technology and are likely to be applied to future genetic technologies, including gene drives. Furthermore, a mistrust of government regulatory agencies, the publication of scientific reports claiming adverse effects of GE crops, the involvement of corporations as the first GE crop developers, the lack of identifiable consumer benefit, and low public understanding of the technology further contributed to the lack of acceptance. Coupled with more emotionally impactful messaging to the public by opposition groups and the general tendency of negative messages to be more credible than positive ones, GE crops failed to gain a place in European agriculture, thus influencing African acceptance and government policy. From this experience, the following lessons have been learned that would apply to the deployment of gene drives, in Africa:

It will be important to establish trust in those who are developing the technology, as well as in those who are making regulatory decisions. Engagement of the community, where those who are involved are able to make genuine contributions to the decision-making process, are necessary to achieve that trust. The use of tools to facilitate participatory modeling could be considered in order to enhance current community engagement efforts.

Trusted, accurate information on gene drives should be made available to the general public, journalists, and scientists who are not connected with the field. Those sources of information should also be able to summarize and analyze important scientific results and emerging issues in the field in order to place those developments in the proper context. Engagement should involve more opportunities for participation of stakeholders in conceptualizing, planning, and decision-making.

Diversifying the source of funding for gene drive research and development, particularly by participation of countries and regional bodies, would show that country or regional interests are represented.

Efforts by developers and neutral groups to provide the public and decisionmakers with a more thorough understanding of the benefits and risks of this technology, especially to local communities, would help them reach more informed decisions.

A better understanding of gene drive technology can be fostered by governments, as part of established biosafety policy in several African countries. Developers and neutral groups could also be helpful in increasing public understanding of the technology of genetic engineering, including gene drives.

Effective messaging to balance the messaging of groups opposed to gene drives is needed. These messages should be not only factual but also have emotional and intuitive appeal.

     

Loss of RecA function affects the ability of Escherichia coli to maintain recombinant plasmids containing a Ter site.

In the Escherichia coli chromosome, DNA replication forks arrested by a Tus-Ter complex or by DNA damage are reinitiated through pathways that involve RecA and numerous other recombination functions. To examine the role of recombination in the processing of replication forks arrested by a Tus-Ter complex, the requirements for recombination-associated gene products were assessed in cells carrying Ter plasmids, i.e., plasmids that contain a Ter site oriented to block DNA replication. Of the E. coli recombination functions tested, only loss of recA conferred an observable phenotype on cells containing a Ter plasmid, which was inefficient transformation and reduced ability to maintain a Ter plasmid when Tus was expressed. Given the current understanding of replication reinitiation, the simplest explanation for the restriction of Ter plasmid maintenance was a reduced ability to restart plasmid replication in a recA tus(+) background. However, we were unable to detect a difference in the efficiency of replication arrest by Tus in recA-proficient and recA-deficient cells, which suggests that the inability to restart arrested replication forks is not the cause of the restriction on growth, but is due to an additional function provided by RecA. Other explanations for restriction of Ter plasmid maintenance were examined, including plasmid multimerization, plasmid rearrangements, and copy number differences. The most likely cause of the restriction on Ter plasmid maintenance was a reduced copy number in recA cells that was detected when the copy number was measured in relation to an external control. Possibly, loss of RecA function leads to improper processing of replication forks arrested at a Ter site, leading to the generation of degradation-prone substrates.     

Lessons from Kitty Hawk: from feasibility to routine clinical use for the field of proteomic pattern diagnostics

Proteomic pattern diagnostics is a rapidly evolving field of science. Despite the increasingly large number of laboratories reporting exciting success with this concept, recent speculation concerning reproducibility and the nature and identities of the information content of the pattern constituents have served to defocus and polarize the community. These controversies will be rendered obsolete as the field accelerates into a new realm of clinical diagnostics. This new era will see the currently dry biomarker pipeline flooded with new candidate molecules, and the mass spectrometer will continue its maturation into a dominant clinical platform. We reflect on the important lessons gleaned from the Wright brothers' attempts at controlled heavier-than-air flight as a model for perseverance and a view to the very near future for proteomic pattern diagnostics.     

From sustainability to well-being: Lessons learned from the use of sustainable development indicators at national and EU level

Indicators describing sustainability and, more recently, well-being have raised considerable interest throughout the world. Much conceptual and empirical research exists focusing on the criteria for sustainability and development of indicators, while relatively few studies examine the actual use and influence of indicators. Employing document analysis and interviews of key actors, we explore the use of sustainable development indicators at national and EU level and draw forth lessons relevant for topical discussion of the measurement of human well-being. We apply a conceptual model of three main types of indicator use: instrumental, conceptual, and political. The results indicate that the use of sustainability indicators is mainly confined to the ‘indicator circuit’ formed by indicator-developers themselves and actors obliged to use the indicators. The results suggest that direct instrumental use of indicators shows limited potential, whereas conceptual use is the key for enhanced indicator influence in the long term. Political use of indicators cannot be controlled by the indicator-developers.     

Lessons learned developing a diagnostic tool for HIV-associated dementia feasible to implement in resource-limited settings: pilot testing in Kenya

Objective

To conduct a preliminary evaluation of the utility and reliability of a diagnostic tool for HIV-associated dementia (HAD) for use by primary health care workers (HCW) which would be feasible to implement in resource-limited settings.

Background

In resource-limited settings, HAD is an indication for anti-retroviral therapy regardless of CD4 T-cell count. Anti-retroviral therapy, the treatment for HAD, is now increasingly available in resource-limited settings. Nonetheless, HAD remains under-diagnosed likely because of limited clinical expertise and availability of diagnostic tests. Thus, a simple diagnostic tool which is practical to implement in resource-limited settings is an urgent need.

Methods

A convenience sample of 30 HIV-infected outpatients was enrolled in Western Kenya. We assessed the sensitivity and specificity of a diagnostic tool for HAD as administered by a primary HCW. This was compared to an expert clinical assessment which included examination by a physician, neuropsychological testing, and in selected cases, brain imaging. Agreement between HCW and an expert examiner on certain tool components was measured using Kappa statistic.

Results

The sample was 57% male, mean age was 38.6 years, mean CD4 T-cell count was 323 cells/µL, and 54% had less than a secondary school education. Six (20%) of the subjects were diagnosed with HAD by expert clinical assessment. The diagnostic tool was 63% sensitive and 67% specific for HAD. Agreement between HCW and expert examiners was poor for many individual items of the diagnostic tool (K = .03–.65). This diagnostic tool had moderate sensitivity and specificity for HAD. However, reliability was poor, suggesting that substantial training and formal evaluations of training adequacy will be critical to enable HCW to reliably administer a brief diagnostic tool for HAD.     

How to use wind erosion to restore and maintain the inland drift-sand ecotype in the Netherlands?

Dutch inland drift-sands are of great value to nature and house several Red-Listed species unique for Europe. The inland drift-sand landscape consists of three different intertwined wind erosion zones. Together they form the conditions where a mosaic of vegetation at different stages of development can thrive. Under present environmental conditions these drift-sands stabilise and tend to disappear. So far there is a lack of a management strategy to maintain the drift-sands after restoration. This paper presents a new management concept based on process management. The main objective of our new management strategy is to maintain optimum field conditions for wind erosion in a manner and scale that allow it to act as a landscape differentiating process. This process management strategy consists of: (1) an overall management plan for a period of 10–15 years; and (2) a short-term (1–3 years) maintenance plan to keep the erosion potential in the selected deflation zones sufficiently high. Details of our general management strategy such as the potential of drift-sand areas, the alternative measures to be undertaken, economic and social considerations and the monitoring and evaluation of our process management approach are elaborated in separate sections. Process management with scheduled maintenance will result in a more stable drift-sand habitat in which the typical drift-sand vegetation and fauna species are better able to survive. In other words, the answer to the question ‘How to use wind erosion to restore and maintain the inland drift-sand ecotype?’ is blowing in the wind if we maintain the optimum conditions for wind erosion.