A phage tubulin assembles dynamic filaments by an atypical mechanism to center viral DNA within the host cell

Tubulins are essential for the reproduction of many eukaryotic viruses, but historically, bacteriophage were assumed not to require a cytoskeleton. Here, we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with an unusual, extended C terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in-vitro-formed filaments, indicating how interactions between the C terminus and the following monomer drive polymerization. PhuZ forms a filamentous array that is required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. Thus, we show that PhuZ assembles a spindle-like array that functions analogously to the microtubule-based spindles of eukaryotes.     

Traumatic Wound Microbiome Workshop

On May 9-10, 2011, the Walter Reed Army Institute of Research, as the Army Center of Excellence for Infectious Disease, assembled over a dozen leaders in areas related to research into the communities of microorganisms which colonize and infect traumatic wounds. The objectives of the workshop were to obtain guidance for government researchers, to spur research community involvement in the field of traumatic wound research informed by a microbiome perspective, and to spark collaborative efforts serving the Wounded Warriors and similarly wounded civilians. During the discussions, it was made clear that the complexity of these infections will only be met by developing a new art of clinical practice that engages the numerous microbes and their ecology. It requires the support of dedicated laboratories and technologists who advance research methods such as community sequencing, as well as the kinds of data analysis expertise and facilities. These strategies already appear to be bearing fruit in the clinical management of chronic wounds. There are now funding announcements and programs supporting this area of research open to extramural collaborators.     

Response to "The anglerfish deception"

The correspondents argue that “The anglerfish deception” contains omissions, errors, misunderstandings and misinterpretations.EMBO reports (2012) advanced online publication; doi: 10.1038/embor.2012.71EMBO reports (2012) 13 2, 100–105; doi: 10.1038/embor.2011.254The commentary [1] on aspects of genetically modified organism (GMO) regulation, risk assessment and risk management in the EU contains omissions, errors, misunderstandings and misinterpretations. As background, environmental risk assessment (ERA) of genetically modified (GM) plants for cultivation in the EU is conducted by applicants following principles and data requirements described in the Guidance Document (ERA GD) established by the European Food Safety Authority (EFSA) [2], which follows the tenets of Directive 2001/18/EC. The ERA GD was not referenced in [1], which wrongly referred only to EFSA guidance that does not cover ERA. Applications for cultivation of a GM plant containing the ERA, submitted to the European Commission (EC), are checked by the EFSA to ensure they address all the requirements specified in its ERA GD [2]. A lead Member State (MS) is then appointed to conduct the initial evaluation of the application, requesting further information from the applicant if required. The MS evaluation is forwarded to the EC, EFSA and all other MSs. Meanwhile, all other MSs can comment on the application and raise concerns. The EFSA GMO Panel carefully considers the content of the application, the lead MS Opinion, other MSs'' concerns, all relevant data published in the scientific literature, and the applicant''s responses to its own requests for further information. The Panel then delivers its Opinion on the application, which covers all the potential environmental areas of risk listed in 2001/18/EC. This Opinion is sent to the EC, all MSs and the applicant and published in the EFSA journal (efsa.europa.eu). Panel Opinions on GM plants for cultivation consider whether environmental harm might be caused, and, if so, suggest possible management to mitigate these risks, and make recommendations for post-market environmental monitoring (PMEM). The final decision on whether to allow the cultivation of GM plants, and any specific conditions for management and monitoring, rests with the EC and MSs and is not within the remit of the EFSA.Against this background we respond to several comments in [1]. Regarding the Comparative Safety Assessment of GM plants and whether or not further questions are asked following this assessment, the Comparative Safety Assessment, described fully in [2], is not a ‘first step''. It is a general principle that forms a central part of the ERA process, as introduced in section 2.1 of [2]. Each ERA starts with problem formulation and identification, facilitating a structured approach to identifying potential risks and scientific uncertainties; following this critical first step many further questions must be asked and addressed. In [2] it is clearly stated that all nine specific areas of risk listed in 2001/18/EC must be addressed—persistence and invasiveness; vertical gene flow; horizontal gene flow; interactions with target organisms; interactions with non-target organisms; human health; animal health; biogeochemical processes; cultivation, management and harvesting techniques. Under the Comparative Safety Assessment, following problem formulation, each of these areas of risk must be assessed by using a six-step approach, involving hazard identification, hazard characterization, exposure assessment, risk characterization, risk management strategies and an overall risk evaluation and conclusion. Indeed, far from asking “no further questions” [1], the EFSA GMO Panel always sends a sequence of written questions to the applicant as part of the ERA process to achieve a complete set of data to support the ERA evaluation (on average about ten per application).The principle of comparative analysis in ERA—sometimes referred to as substantial equivalence in the risk assessment of food and feed—is not discredited. The comparative approach is supported by all of the world''s leading national science academies [for example, 3]; none has recommended an alternative. The principle is enshrined in risk assessment guidelines issued by all relevant major international bodies, including the World Health Organization, the Food and Agriculture Organization of the United Nations and the Organisation for Economic Co-operation and Development. Critics of this approach have failed to propose any credible alternative baseline to risk assess GMOs. The comparative analysis as described in [2] is not a substitute for a safety assessment, but is a tool within the ERA [4] through which comparisons are made with non-GM counterparts in order to identify hazards associated with the GM trait, the transformation process and the associated management systems, which are additional to those impacts associated with the non-GM plant itself. The severity and frequency of these hazards are then quantified in order to assess the levels of risks associated with the novel features of the GM plant and its cultivation.European Parliament (EP) communications include that “the characteristics of the receiving environments and the geographical areas in which GM plants may be cultivated should be duly taken into account”. We agree, and the ERA GD [2] recognizes explicitly that receiving environments differ across the EU, and that environmental impacts might differ regionally. Therefore, the ERA GD [2] demands that such differences be fully accounted for in cultivation applications and that receiving environments be assessed separately in each of the nine specific areas of risk (see section 2.3.2). Furthermore, [2] states in section 3.5 that the ERA should consider scenarios representative of the diversity of situations that might occur and assess their potential implications. The EP communications state that “the long-term environmental effects of GM crops, as well as their potential effects on non-target organisms, should be rigorously assessed”. This is covered explicitly in section 2.3.4 of [2], and developed in the recent guidance on PMEM [5].The EFSA is committed to openness, transparency and dialogue and meets regularly with a wide variety of stakeholders including non-governmental organizations (NGOs) [6] to discuss GMO topics. That the EFSA is neither a centralized nor a singular voice of science in the EU is clear, because the initial report on the ERA is delivered by a MS, not the EFSA; all MSs can comment on the ERA; and EFSA GMO Panel Opinions respond transparently to every concern raised by each MS. Following publication, the EFSA regularly attends the SCFCAH Committee (comprising MS representatives) to account for its Opinions. The involvement of all MSs in the evaluation process ensures that concerns relating to their environments are addressed in the ERA. Subsequently, MSs can contribute to decisions on the management and monitoring of GM plants in their territories if cultivation is approved.In recent years, several MSs have used the ‘safeguard clause'', Article 23 of 2001/18/EC, to attempt to ban the cultivation of specific GM plants in their territories, despite earlier EFSA Panel Opinions on those plants. But the claim that “the risk science of the EFSA''s GM Panel has been publicly disputed in Member State''s justifications of their Article 23 prohibitions” needs to be placed into context [1]. When a safeguard clause (SC) is issued by a MS, the EFSA GMO Panel is often asked by the EC to deliver an Opinion on the scientific basis of the SC. The criteria on which to judge the documentation accompanying a SC are whether: (i) it represents new scientific evidence—and is not just repetition of information previously assessed—that demonstrates a risk to human and animal health and the environment; and (from the guidance notes to Annex II of 2001/18/EC) (ii) it is proportionate to the level of risk and to the level of uncertainty. It is pertinent that on 8 September 2011, the EU Court of Justice ruled that ‘with a view to the adoption of emergency measures, Article 34 of Regulation (EC) No 1829/2003 requires Member States to establish, in addition to urgency, the existence of a situation which is likely to constitute a clear and serious risk to human health, animal health or the environment''. Scientific literature is monitored continually by the Panel and relevant new work is examined to determine whether it raises any new safety concern. In all cases where the EFSA was consulted by the EC, there has been no new scientific information presented that would invalidate the Panel''s previous assessment.Throughout [1] the text demonstrates a fundamental misunderstanding of the distinction between ERA and risk management. ERA is the responsibility of the EFSA, although it is asked for its opinion on risk management methodology by the EC. Risk management implementation is the responsibility of the EC and MSs. Hence, the setting of protection goals is an issue for risk managers and might vary between MSs. However, the ERA GD [2], through its six-step approach, makes it mandatory for applications to relate the results of any studies directly to limits of environmental concern that reflect protection goals and the level of change deemed acceptable. Indeed, the recent EFSA GMO Panel Opinions on Bt-maize events [for example, 7] have been written specifically to provide MSs and risk managers with the tools to adapt the results of the quantified ERA to their own local protection goals. This enables MSs to implement risk management and PMEM proportional to the risks identified in their territories.The EFSA GMO Panel comprises independent researchers, appointed for their expertise following an open call to the scientific community. The Panel receives able support from staff of the EFSA GMO Unit and numerous ad hoc members of its working groups. It has no agenda and is neither pro- or anti-GMOs; its paramount concern is the quality of the science underpinning its Guidance Documents and Opinions.     

Formin leaky cap allows elongation in the presence of tight capping proteins

Formins, characterized by formin homology domains FH1 and FH2, are required to assemble certain F-actin structures including actin cables, stress fibers, and the contractile ring. FH1FH2 in a recombinant fragment from a yeast formin (Bni1p) nucleates actin filaments in vitro. It also binds to the filament barbed end where it appears to act as a "leaky" capper, slowing both polymerization and depolymerization by approximately 50%. We now find that FH1FH2 competes with tight capping proteins (including gelsolin and heterodimeric capping protein) for the barbed end. We also find that FH1FH2 forms a tetramer. The observation that this formin protects an end from capping but still allows elongation confirms that it is a leaky capper. This is significant because a nucleator that protects a new barbed end from tight cappers will increase the duration of elongation and thus the total amount of F-actin. The ability of FH1FH2 to dimerize probably allows the formin to walk processively with the barbed end as the filament elongates.     

A kinase-independent function of PAK is crucial for pathogen-mediated actin remodelling

The p21-activated kinase (PAK) family regulate a multitude of cellular processes, including actin cytoskeleton remodelling. Numerous bacterial pathogens usurp host signalling pathways that regulate actin reorganisation in order to promote Infection. Salmonella and pathogenic Escherichia coli drive actin-dependent forced uptake and intimate attachment respectively. We demonstrate that the pathogen-driven generation of both these distinct actin structures relies on the recruitment and activation of PAK. We show that the PAK kinase domain is dispensable for this actin remodelling, which instead requires the GTPase-binding CRIB and the central poly-proline rich region. PAK interacts with and inhibits the guanine nucleotide exchange factor β-PIX, preventing it from exerting a negative effect on cytoskeleton reorganisation. This kinase-independent function of PAK may be usurped by other pathogens that modify host cytoskeleton signalling and helps us better understand how PAK functions in normal and diseased eukaryotic cells.     

Viral clearance using disposable systems in monoclonal antibody commercial downstream processing

Once highly selective protein A affinity is chosen for robust mAb downstream processing, the major role of polishing steps is to remove product related impurities, trace amounts of host cell proteins, DNA/RNA, and potential viral contaminants. Disposable systems can act as powerful options either to replace or in addition to polishing column chromatography to ensure product purity and excellent viral clearance power for patients' safety. In this presentation, the implementation of three disposable systems such as depth filtration, membrane chromatography, and nanometer filtration technology in a commercial process are introduced. The data set of viral clearance with these systems is presented. Application advantages and disadvantages including cost analysis are further discussed.     

Order-disorder-order transitions mediate the activation of cholera toxin

Cholera toxin (CT) holotoxin must be activated to intoxicate host cells. This process requires the intracellular dissociation of the enzymatic CTA1 domain from the holotoxin components CTA2 and B5, followed by subsequent interaction with the host factor ADP ribosylation factor 6 (ARF6)-GTP. We report the first NMR-based solution structural data for the CT enzymatic domain (CTA1). We show that this free enzymatic domain partially unfolds at the C-terminus and binds its protein partners at both the beginning and the end of this activation process. Deviations from random coil chemical shifts (Δδ_coil) indicate helix formation in the activation loop, which is essential to open the toxin's active site and occurs prior to its association with human protein ARF6. We performed NMR titrations of both free CTA1 and an active CTA1:ARF6-GTP complex with NAD⁺, which revealed that the formation of the complex does not significantly enhance NAD⁺ binding. Partial unfolding of CTA1 is further illustrated by using 4,4′-bis(1-anilinonaphthalene 8-sulfonate) fluorescence as an indicator of the exposed hydrophobic character of the free enzyme, which is substantially reduced when bound to ARF6-GTP. We propose that the primary role of ARF6's allostery is to induce refolding of the C-terminus of CTA1. Thus, as a folded globular toxin complex, CTA1 escapes the chaperone and proteasomal components of the endoplasmic reticulum associated degradation pathway in the cytosol and then proceeds to ADP ribosylate its target G_sα, triggering the downstream events associated with the pathophysiology of cholera.     

Effect of cell polarization on hepatitis C virus entry

The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.