Thomas Rudel

Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication

Influenza A virus, being responsible for seasonal epidemics and reoccurring pandemics, represents a worldwide threat to public health. High mutation rates facilitate the generation of viral escape mutants, rendering vaccines and drugs directed against virus-encoded targets potentially ineffective. In contrast, targeting host cell determinants temporarily dispensable for the host but crucial for virus replication could prevent viral escape. Here we report the discovery of 287 human host cell genes influencing influenza A virus replication in a genome-wide RNA interference (RNAi) screen. Using an independent assay we confirmed 168 hits (59%) inhibiting either the endemic H1N1 (119 hits) or the current pandemic swine-origin (121 hits) influenza A virus strains, with an overlap of 60%. Notably, a subset of these common hits was also essential for replication of a highly pathogenic avian H5N1 strain. In-depth analyses of several factors provided insights into their infection stage relevance. Notably, SON DNA binding protein (SON) was found to be important for normal trafficking of influenza virions to late endosomes early in infection. We also show that a small molecule inhibitor of CDC-like kinase 1 (CLK1) reduces influenza virus replication by more than two orders of magnitude, an effect connected with impaired splicing of the viral M2 messenger RNA. Furthermore, influenza-virus-infected p27-/- (cyclin-dependent kinase inhibitor 1B; Cdkn1b) mice accumulated significantly lower viral titres in the lung, providing in vivo evidence for the importance of this gene. Thus, our results highlight the potency of genome-wide RNAi screening for the dissection of virus–host interactions and the identification of drug targets for a broad range of influenza viruses.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Changing drivers of deforestation and new opportunities for conservation.

Over the past 50 years, human agents of deforestation have changed in ways that have potentially important implications for conservation efforts. We characterized these changes through a meta-analysis of case studies of land-cover change in the tropics. From the 1960s to the 1980s, small-scale farmers, with state assistance, deforested large areas of tropical forest in Southeast Asia and Latin America. As globalization and urbanization increased during the 1980s, the agents of deforestation changed in two important parts of the tropical biome, the lowland rainforests in Brazil and Indonesia. Well-capitalized ranchers, farmers, and loggers producing for consumers in distant markets became more prominent in these places and this globalization weakened the historically strong relationship between local population growth and forest cover. At the same time, forests have begun to regrow in some tropical uplands. These changing circumstances, we believe, suggest two new and differing strategies for biodiversity conservation in the tropics, one focused on conserving uplands and the other on promoting environmental stewardship in lowlands and other areas conducive to industrial agriculture.

Agricultural intensification and changes in cultivated areas, 1970–2005

Does the intensification of agriculture reduce cultivated areas and, in so doing, spare some lands by concentrating production on other lands? Such sparing is important for many reasons, among them the enhanced abilities of released lands to sequester carbon and provide other environmental services. Difficulties measuring the extent of spared land make it impossible to investigate fully the hypothesized causal chain from agricultural intensification to declines in cultivated areas and then to increases in spared land. We analyze the historical circumstances in which rising yields have been accompanied by declines in cultivated areas, thereby leading to land-sparing. We use national-level United Nations Food and Agricultural Organization data on trends in cropland from 1970–2005, with particular emphasis on the 1990–2005 period, for 10 major crop types. Cropland has increased more slowly than population during this period, but paired increases in yields and declines in cropland occurred infrequently, both globally and nationally. Agricultural intensification was not generally accompanied by decline or stasis in cropland area at a national scale during this time period, except in countries with grain imports and conservation set-aside programs. Future projections of cropland abandonment and ensuing environmental services cannot be assumed without explicit policy intervention.

A Contemporary Assessment of Change in Humid Tropical Forests

In recent decades the rate and geographic extent of land-use and land-cover change has increased throughout the worlds humid tropical forests. The pan-tropical geography of forest change is a challenge to assess, and improved estimates of the human footprint in the tropics are critical to understanding potential changes in biodiversity. We combined recently published and new satellite observations, along with images from Google Earth and a literature review, to estimate the contemporary global extent of deforestation, selective logging, and secondary regrowth in humid tropical forests. Roughly 1.4% of the biome was deforested between 2000 and 2005. As of 2005, about half of the humid tropical forest biome contained 50% or less tree cover. Although not directly comparable to deforestation, geographic estimates of selective logging indicate that at least 20% of the humid tropical forest biome was undergoing some level of timber harvesting between 2000 and 2005. Forest recovery estimates are even less certain, but a compilation of available reports suggests that at least 1.2% of the humid tropical forest biome was in some stage of long-term secondary regrowth in 2000. Nearly 70% of the regrowth reports indicate forest regeneration in hilly, upland, and mountainous environments considered marginal for large-scale agriculture and ranching. Our estimates of the human footprint are conservative because they do not resolve very small-scale deforestation, low-intensity logging, and unreported secondary regrowth, nor do they incorporate other impacts on tropical forest ecosystems, such as fire and hunting. Our results highlight the enormous geographic extent of forest change throughout the humid tropics and the considerable limitations of the science and technology available for such a synthesis.

Forest transitions, trade, and the global displacement of land use

Reducing tropical deforestation is an international priority, given its impacts on carbon emissions and biodiversity. We examined whether recent forest transitions—a shift from net deforestation to net reforestation—involved a geographic displacement of forest clearing across countries through trade in agricultural and forest products. In most of the seven developing countries that recently experienced a forest transition, displacement of land use abroad accompanied local reforestation. Additional global land-use change embodied in their net wood trade offset 74% of their total reforested area. Because the reforesting countries continued to export more agricultural goods than they imported, this net displacement offset 22% of their total reforested area when both agriculture and forestry sectors are included. However, this net displacement increased to 52% during the last 5 y. These countries thus have contributed to a net global reforestation and/or decrease in the pressure on forests, but this global environmental benefit has been shrinking during recent years. The net decrease in the pressure on forests does not account for differences in their ecological quality. Assessments of the impacts of international policies aimed at reducing global deforestation should integrate international trade in agricultural and forest commodities.

Prohibitin is required for Ras-induced Raf–MEK–ERK activation and epithelial cell migration

Ras proteins control the signalling pathways that are responsible for normal growth and malignant transformation. Raf protein kinases are direct Ras effector proteins that initiate the mitogen-activated protein kinase (MAPK) cascade, which mediates diverse biological functions such as cell growth, survival and differentiation. Here we show that prohibitin, a ubiquitously expressed and evolutionarily conserved protein is indispensable for the activation of the Raf–MEK–ERK pathway by Ras. The membrane targeting and activation of C-Raf by Ras needs prohibitin in vivo. In addition, direct interaction with prohibitin is required for C-Raf activation. C-Raf kinase fails to interact with the active Ras induced by epidermal growth factor in the absence of prohibitin. Moreover, in prohibitin-deficient cells the adhesion complex proteins cadherin and β-catenin relocalize to the plasma membrane and thereby stabilize adherens junctions. Our data show an unexpected role of prohibitin in the activation of the Ras–Raf signalling pathway and in modulating epithelial cell adhesion and migration.

A Tropical Forest Transition? Agricultural Change, Out-migration, and Secondary Forests in the Ecuadorian Amazon

Could old colonization zones in the urbanizing and industrializing countries of Latin America become sites for a tropical forest transition in which reforestation becomes more prevalent than deforestation? We try to answer this question through a case study of land-use change and migration since 1985 in a long-settled region of the Ecuadorian Amazon. Data from remote sensing analyses, household surveys, and land-use maps of individual farms reveal two disparate patterns of reforestation in the region, one on peripheral lands far from roads and the other on lands close to roads. The former pattern characterizes most places experiencing a forest transition; the latter pattern does not. Roadside reforestation has occurred in part because Amerindian smallholders have abandoned cattle ranching in order to practice short-cycle shifting cultivation of crops for expanding urban and export markets. This example suggests that tropical forest transitions may differ from earlier temperate forest transitions in that reforestation does not signify land abandonment. Even as they come to rely more completely on nonfarm sources of income, smallholders in developing countries continue to manage their land, reforesting when it eliminates expenses or promises new, near-term streams of income.

MicroRNA profiling of clear cell renal cell cancer identifies a robust signature to define renal malignancy

MicroRNAs are short single‐stranded RNAs that are associated with gene regulation at the transcriptional and translational level. Changes in their expression were found in a variety of human cancers. Only few data are available on microRNAs in clear cell renal cell carcinoma (ccRCC). We performed genome‐wide expression profiling of microRNAs using microarray analysis and quantification of specific microRNAs by TaqMan real‐time RT‐PCR. Matched malignant and non‐malignant tissue samples from two independent sets of 12 and 72 ccRCC were profiled. The microarray‐based experiments identified 13 over‐expressed and 20 down‐regulated microRNAs in malignant samples. Expression in ccRCC tissue samples compared with matched non‐malignant samples measured by RT‐PCR was increased on average by 2.7‐ to 23‐fold for the hsa‐miR‐16, −452*, −224, −155 and −210, but decreased by 4.8‐ to 138‐fold for hsa‐miR‐200b, −363, −429, −200c, −514 and −141. No significant associations between these differentially expressed microRNAs and the clinico‐pathological factors tumour stage, tumour grade and survival rate were found. Nevertheless, malignant and non‐malignant tissue could clearly be differentiated by their microRNA profile. A combination of miR‐141 and miR‐155 resulted in a 97% overall correct classification of samples. The presented differential microRNA pattern provides a solid basis for further validation, including functional studies.

Neisserial porin (PorB) causes rapid calcium influx in target cells and induces apoptosis by the activation of cysteine proteases

The porin (PorB) of Neisseria gonorrhoeae is an intriguing bacterial factor owing to its ability to translocate from the outer bacterial membrane into host cell membranes where it modulates the infection process. Here we report on the induction of programmed cell death after prolonged infection of epithelial cells with pathogenic Neisseria species. The underlying mechanism we propose includes translocation of the porin, a transient increase in cytosolic Ca2+ and subsequent activation of the Ca2+ dependent protease calpain as well as proteases of the caspase family. Blocking the porin channel by ATP eliminates the Ca2+ signal and also abolishes its pro‐apoptotic function. The neisserial porins share structural and functional homologies with the mitochondrial voltage‐dependent anion channels (VDAC). The neisserial porin may be an analogue or precursor of the ancient permeability transition pore, the putative central regulator of apoptosis.