Functional analysis and structural modeling of human APOBEC3G reveal the role of evolutionarily conserved elements in the inhibition of human immunodeficiency virus type 1 infection and Alu transposition.

Retroelements are important evolutionary forces but can be deleterious if left uncontrolled. Members of the human APOBEC3 family of cytidine deaminases can inhibit a wide range of endogenous, as well as exogenous, retroelements. These enzymes are structurally organized in one or two domains comprising a zinc-coordinating motif. APOBEC3G contains two such domains, only the C terminal of which is endowed with editing activity, while its N-terminal counterpart binds RNA, promotes homo-oligomerization, and is necessary for packaging into human immunodeficiency virus type 1 (HIV-1) virions. Here, we performed a large-scale mutagenesis-based analysis of the APOBEC3G N terminus, testing mutants for (i) inhibition of vif-defective HIV-1 infection and Alu retrotransposition, (ii) RNA binding, and (iii) oligomerization. Furthermore, in the absence of structural information on this domain, we used homology modeling to examine the positions of functionally important residues and of residues found to be under positive selection by phylogenetic analyses of primate APOBEC3G genes. Our results reveal the importance of a predicted RNA binding dimerization interface both for packaging into HIV-1 virions and inhibition of both HIV-1 infection and Alu transposition. We further found that the HIV-1-blocking activity of APOBEC3G N-terminal mutants defective for packaging can be almost entirely rescued if their virion incorporation is forced by fusion with Vpr, indicating that the corresponding region of APOBEC3G plays little role in other aspects of its action against this pathogen. Interestingly, residues forming the APOBEC3G dimer interface are highly conserved, contrasting with the rapid evolution of two neighboring surface-exposed amino acid patches, one targeted by the Vif protein of primate lentiviruses and the other of yet-undefined function.

NanoImpactNet - building a multi-stakeholder dialogue on the health and environmental impact of nanomaterials

NanoImpactNet (NIN) is a multidisciplinary European Commission funded network on the environmental, health and safety (EHS) impact of nanomaterials. The 24 founding scientific institutes are leading European research groups active in the fields of nanosafety, nanorisk assessment and nanotoxicology. This 4-year project is the new focal point for information exchange within the research community. Contact with other stakeholders is vital and their needs are being surveyed. NIN is communicating with 100s of stakeholders: businesses; internet platforms; industry associations; regulators; policy makers; national ministries; international agencies; standard-setting bodies and NGOs concerned by labour rights, EHS or animal welfare. To improve this communication, internet research, a questionnaire distributed via partners and targeted phone calls were used to identify stakeholders' interests and needs. Knowledge gaps and the necessity for further data mentioned by representatives of all stakeholder groups in the targeted phone calls concerned: • the potential toxic and safety hazards of nanomaterials throughout their lifecycles; • the fate and persistence of nanoparticles in humans, animals and the environment; • the associated risks of nanoparticle exposure; • greater participation in: the preparation of nomenclature, standards, methodologies, protocols and benchmarks; • the development of best practice guidelines; • voluntary schemes on responsibility; • databases of materials, research topics and themes, but also of expertise. These findings suggested that stakeholders and NIN researchers share very similar knowledge needs, and that open communication and free movement of knowledge will benefit both researchers and industry. Subsequently a workshop was organised by NIN focused on building a sustainable multi-stakeholder dialogue. Specific questions were asked to different stakeholder groups to encourage discussions and open communication. 1. What information do stakeholders need from researchers and why? The discussions about this question confirmed the needs identified in the targeted phone calls. 2. How to communicate information? While it was agreed that reporting should be enhanced, commercial confidentiality and economic competition were identified as major obstacles. It was recognised that expertise was needed in the areas of commercial law and economics for a wellinformed treatment of this communication issue. 3. Can engineered nanomaterials be used safely? The idea that nanomaterials are probably safe because some of them have been produced 'for a long time', was questioned, since many materials in common use have been proved to be unsafe. The question of safety is also about whether the public has confidence. New legislation like REACH could help with this issue. Hazards do not materialise if exposure can be avoided or at least significantly reduced. Thus, there is a need for information on what can be regarded as acceptable levels of exposure. Finally, it was noted that there is no such thing as a perfectly safe material but only boundaries. At this moment we do not know where these boundaries lie. The matter of labelling of products containing nanomaterials was raised, as in the public mind safety and labelling are connected. This may need to be addressed since the issue of nanomaterials in food, drink and food packaging may be the first safety issue to attract public and media attention, and this may have an impact on 'nanotechnology as a whole. 4. Do we need more or other regulation? Any decision making process should accommodate the changing level of uncertainty. To address the uncertainties, adaptations of frameworks such as REACH may be indicated for nanomaterials. Regulation is often needed even if voluntary measures are welcome because it mitigates the effects of competition between industries. Data cannot be collected on voluntary bases for example. NIN will continue with an active stakeholder dialogue to further build on interdisciplinary relationships towards a healthy future with nanotechnology.

Regulatory assessment of in vitro skin corrosion and irritation data within the European framework: Workshop recommendations

Validated in vitro methods for skin corrosion and irritation were adopted by the OECD and by the European Union during the last decade. In the EU, Switzerland and countries adopting the EU legislation, these assays may allow the full replacement of animal testing for identifying and classifying compounds as skin corrosives, skin irritants, and non irritants. In order to develop harmonised recommendations on the use of in vitro data for regulatory assessment purposes within the European framework, a workshop was organized by the Swiss Federal Office of Public Health together with ECVAM and the BfR. It comprised stakeholders from various European countries involved in the process from in vitro testing to the regulatory assessment of in vitro data. Discussions addressed the following questions: (1) the information requirements considered useful for regulatory assessment; (2) the applicability of in vitro skin corrosion data to assign the corrosive subcategories as implemented by the EU Classification, Labelling and Packaging Regulation; (3) the applicability of testing strategies for determining skin corrosion and irritation hazards; and (4) the applicability of the adopted in vitro assays to test mixtures, preparations and dilutions. Overall, a number of agreements and recommendations were achieved in order to clarify and facilitate the assessment and use of in vitro data from regulatory accepted methods, and ultimately help regulators and scientists facing with the new in vitro approaches to evaluate skin irritation and corrosion hazards and risks without animal data.

Angiogenesis in Wounds Treated by Microdeformational Wound Therapy.

BACKGROUND:: Mechanical forces play an important role in tissue neovascularization and are a constituent part of modern wound therapies. The mechanisms by which vacuum assisted closure (VAC) modulates wound angiogenesis are still largely unknown. OBJECTIVE:: To investigate how VAC treatment affects wound hypoxia and related profiles of angiogenic factors as well as to identify the anatomical characteristics of the resultant, newly formed vessels. METHODS:: Wound neovascularization was evaluated by morphometric analysis of CD31-stained wound cross-sections as well as by corrosion casting analysis. Wound hypoxia and mRNA expression of HIF-1α and associated angiogenic factors were evaluated by pimonidazole hydrochloride staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Vascular endothelial growth factor (VEGF) protein levels were determined by western blot analysis. RESULTS:: VAC-treated wounds were characterized by the formation of elongated vessels aligned in parallel and consistent with physiologically function, compared to occlusive dressing control wounds that showed formation of tortuous, disoriented vessels. Moreover, VAC-treated wounds displayed a well-oxygenated wound bed, with hypoxia limited to the direct proximity of the VAC-foam interface, where higher VEGF levels were found. By contrast, occlusive dressing control wounds showed generalized hypoxia, with associated accumulation of HIF-1α and related angiogenic factors. CONCLUSIONS:: The combination of established gradients of hypoxia and VEGF expression along with mechanical forces exerted by VAC therapy was associated with the formation of more physiological blood vessels compared to occlusive dressing control wounds. These morphological changes are likely a necessary condition for better wound healing.

Cercles de qualité médecins-pharmaciens, pour une responsabilité partagée de la liberté de prescription [Physicians-pharmacists quality circles: shared responsibility of the freedom of prescription].

Physicians-pharmacists quality circles (PPQCs) were introduced in 1997-98 by visionary healthcare practitioners of the French-speaking part of Switzerland with the aim to improve the quality of drug prescription. Indeed the challenge is to manage the 7917 brand names of the Swiss drug market (2010), including 19793 different dosages, galenic formulations and packaging. The impact of these PPQCs on the containment of drug costs and on drug prescribing profiles has been demonstrated and has led to their spread throughout Switzerland. PPQCs provide clear educational benefits and have thus been accredited by various continuous education bodies. In this article, participating physicians and pharmacists share their vision and illustrate how they work and influence the safety and efficiency of drug prescription, a routine process complex enough to warrant sharing of its burden in a constructive interdisciplinary collaboration.

Applications of a transportable Raman spectrometer for the in situ detection of controlled substances at border controls

A transportable Raman spectrometer was tested for the detection of illicit drugs seized during border controls. In a first step, the analysis methodology was optimized using reference substances such as diacetylmorphine (heroin), cocaine and amphetamine (as powder or liquid forms). Adequate focalisation distance and times of analysis, influence of daylight and artificial light sources, repeatability and limits of detection were studied. In a second step the applications and limitations of the technique to detect the illicit substances in different mixtures and containers was evaluated. Transportable Raman spectroscopy was found to be adequate for a rapid screen of liquids and powders for the detection and identification of controlled substances. Additionally, it had the advantage over other portable techniques, such as ion mobility spectrometry, of being non-destructive and capable of rapid analysis of large quantities of substances through containers such as plastic bags and glass bottles.

The hospital pharmacist: an important contributor to improved patient safety in the hospital.

Injectable drugs are high-risk products and their reconstitution in hospital wards is a potential source of errors. Thus, in order to secure the reconstitution process and thereby improve safety, the pharmacy department of Lausanne University Hospital is focusing on developing ready-to-use forms (CIVAS). These preparations are compounded in controlled clean rooms and are analyzed prior to release. In the intensive care unit, amiodarone 12.5 mg/mL in glucose 5% is one of the high-risk preparations, which has led the pharmacy to develop a ready-to-use solution. To this end, a one-year stability study was initiated, and the preliminary results (after six months) are illustrated here. A stability-indicating HPLC method was developed and validated for monitoring the concentration of amiodarone. Batches were stored at 5 °C and 30 °C, which were analyzed immediately after preparation, after one, two, four and six months of storage. The pH and osmolality values were monitored at the respective time intervals. It was observed that after six months, all the results were within specifications. However, the pH values started to decrease after two months when amiodarone was stored at 30 °C. After six months, a degradation peak appeared on the chromatogram of these solutions, which suggested that amiodarone is more stable at 5 °C. The preliminary results obtained in this study indicated that injectable amiodarone solutions are stable for six months under refrigerated storage conditions. The study is ongoing.

Convention-cadre pour la lutte antitabac: un instrument puissant de santé publique. [The Framework Convention for Tobacco Control (FCTC): a powerful public health instrument].

The Framework Convention on Tobacco Control (FCTC) isa global and comprehensive legal framework for reducing demand for tobacco (e.g. price measures; ban on smoking in enclosed places; contents of tobacco products; packaging and labeling; advertising, promotion and sponsorship; liability, tobacco cessation, etc.) and supply (e.g. illicit trade; sales to/by minors, etc.). Adopted in 2003, the FCTC has been ratified by 174 countries so far. Switzerland has signed the treaty in 2004 but ratification will necessitate the implementation of stronger tobacco control measures at the national level. The FCTC is a priority of any strategy to reduce noncommunicable diseases in populations. Broad implementation of the FCTC has the potential to prevent a substantial proportion of the billion of tobacco-related deaths expected in the 21st

Resistance to nucleoside analog reverse transcriptase inhibitors mediated by human immunodeficiency virus type 1 p6 protein.

Resistance of human immunodeficiency virus type 1 (HIV-1) to antiretroviral agents results from target gene mutation within the pol gene, which encodes the viral protease, reverse transcriptase (RT), and integrase. We speculated that mutations in genes other that the drug target could lead to drug resistance. For this purpose, the p1-p6(gag)-p6(pol) region of HIV-1, placed immediately upstream of pol, was analyzed. This region has the potential to alter Pol through frameshift regulation (p1), through improved packaging of viral enzymes (p6(Gag)), or by changes in activation of the viral protease (p6(Pol)). Duplication of the proline-rich p6(Gag) PTAP motif, necessary for late viral cycle activities, was identified in plasma virus from 47 of 222 (21.2%) patients treated with nucleoside analog RT inhibitor (NRTI) antiretroviral therapy but was identified very rarely from drug-naïve individuals. Molecular clones carrying a 3-amino-acid duplication, APPAPP (transframe duplication SPTSPT in p6(Pol)), displayed a delay in protein maturation; however, they packaged a 34% excess of RT and exhibited a marked competitive growth advantage in the presence of NRTIs. This phenotype is reminiscent of the inoculum effect described in bacteriology, where a larger input, or a greater infectivity of an organism with a wild-type antimicrobial target, leads to escape from drug pressure and a higher MIC in vitro. Though the mechanism by which the PTAP region participates in viral maturation is not known, duplication of this proline-rich motif could improve assembly and packaging at membrane locations, resulting in the observed phenotype of increased infectivity and drug resistance.

Model structure of human APOBEC3G.

BACKGROUND: APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) has antiretroviral activity associated with the hypermutation of viral DNA through cytosine deamination. APOBEC3G has two cytosine deaminase (CDA) domains; the catalytically inactive amino-terminal domain of APOBEC3G (N-CDA) carries the Vif interaction domain. There is no 3-D structure of APOBEC3G solved by X-ray or nuclear magnetic resonance. METHODOLOGY/PRINCIPAL FINDINGS: We predicted the structure of human APOBEC3G based on the crystal structure of APOBEC2. To assess the model structure, we evaluated 48 mutants of APOBEC3G N-CDA that identify novel variants altering DeltaVif HIV-1 infectivity and packaging of APOBEC3G. Results indicated that the key residue D128 is exposed at the surface of the model, with a negative local electrostatic potential. Mutation D128K changes the sign of that local potential. In addition, two novel functionally relevant residues that result in defective APOBEC3G encapsidation, R122 and W127, cluster at the surface. CONCLUSIONS/SIGNIFICANCE: The structure model identifies a cluster of residues important for packaging of APOBEC3G into virions, and may serve to guide functional analysis of APOBEC3G.

Determination of bacterial quantity by sonication in vacum-assisted closure (VAC) foams used for treatment of chronic wounds

Background: Negative pressure wound treatment is increasingly used through a Vacuum-Assisted Closure (VAC) device in complex wound situations. For this purpose, sterile polyurethane (PU) and polyvinyl alcohol (PVA) foam dressings are fitted to the wound size and covered with an adhesive drape to create an airtight seal. Little information exists about the type and quantity of microorganisms within the foams. Therefore, we investigated VAC foams after removal from the wound using a validated method (sonication) to detect the bacterial bioburden in the foam consisting as microbial biofilms.Methods: We prospectively included VAC foams (PU and PVA, KCI, Rümlamg, Switzerland) without antibacterial additions (e.g. silver), which were removed from wounds in patients with chronic ulcers from January 2007 through December 2008. Excluded were patients with acute wound infection, necrotizing fasciitis, underlying osteomyelitis or implant. Removed foams from regular changes of dressing were aseptically placed in a container with 100 ml sterile Ringer's solution. Within 4 hours after removal, foams were sonicated for 5 min at 40 kHz (as described in NEJM 2007;357:654). The resulting sonication fluid was cultured at 37°C on aerobic blood agar plates for 5 days. Microbes were quantified as No. of colony-forming units (CFU)/ml sonication fluid and identified to the species level.Results: A total of 68 foams (38 PU and 30 PVA) from 55 patients were included in the study (median age 71 years; range 33-88 years, 57% were man). Foams were removed from the following anatomic sites: sacrum (n=29), ischium (n=18), heel (n=13), calves (n=6) and ankle (n=2). The median duration of being in place was 3 days (range, 1-8 days). In all 68 foams, bacteria were found in large quantities (median 105 CFU/ml, range 102-7 CFU/ml sonication fluid. No differences were found between PU and PVA foams. One type of organisms was found in 11 (16%), two in 17 (24%) and 3 or more in 40 (60%) foams. Gram-negative rods (Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa) were isolated in 70%, followed by Staphylococcus aureus (20%), koagulase-negative staphylococci, streptococci (8%), and enterococci (2%).Conclusion: With sonication, a high density of bacteria present in VAC foams was demonstrated after a median of 3 days. Future studies are needed to investigate whether antimicrobial-impregnated foams can reduce the bacterial load in foams and potentially improve wound healing.

Rapid affinity purification of erythropoietin from biological samples using disposable monoliths.

Identification of post-translational modifications of proteins in biological samples often requires access to preanalytical purification and concentration methods. In the purification step high or low molecular weight substances can be removed by size exclusion filters, and high abundant proteins can be removed, or low abundant proteins can be enriched, by specific capturing tools. In this paper is described the experience and results obtained with a recently emerged and easy-to-use affinity purification kit for enrichment of the low amounts of EPO found in urine and plasma specimens. The kit can be used as a pre-step in the EPO doping control procedure, as an alternative to the commonly used ultrafiltration, for detecting aberrantly glycosylated isoforms. The commercially available affinity purification kit contains small disposable anti-EPO monolith columns (6 ?L volume, Ø7 mm, length 0.15 mm) together with all required buffers. A 24-channel vacuum manifold was used for simultaneous processing of samples. The column concentrated EPO from 20 mL urine down to 55 ?L eluate with a concentration factor of 240 times, while roughly 99.7% of non-relevant urine proteins were removed. The recoveries of Neorecormon (epoetin beta), and the EPO analogues Aranesp and Mircera applied to buffer were high, 76%, 67% and 57%, respectively. The recovery of endogenous EPO from human urine was 65%. High recoveries were also obtained when purifying human, mouse and equine EPO from serum, and human EPO from cerebrospinal fluid. Evaluation with the accredited EPO doping control method based on isoelectric focusing (IEF) showed that the affinity purification procedure did not change the isoform distribution for rhEPO, Aranesp, Mircera or endogenous EPO. The kit should be particularly useful for applications in which it is essential to avoid carry-over effects, a problem commonly encountered with conventional particle-based affinity columns. The encouraging results with EPO propose that similar affinity monoliths, with the appropriate antibodies, should constitute useful tools for general applications in sample preparation, not only for doping control of EPO and other hormones such as growth hormone and insulin but also for the study of post-translational modifications of other low abundance proteins in biological and clinical research, and for sample preparation prior to in vitro diagnostics.

6

Corporations do not exist in a vacuum, they are embedded in a socio-political context. This context shapes our ideas about what managers within corporations and corporations within society do or should do. The rise of a global world order at the threshold of the 21st century is transforming our society at high speed. Questions of business integrity and sustainability move center stage. This book, written on the occasion of the HEC Lausanne centennial by some thirty professors, discusses various aspects of responsible management practices for the 21st century and provides some key insights for managers today and tomorrow.

DNA-DNA interactions in bacteriophage capsids are responsible for the observed DNA knotting.

Recent experiments showed that the linear double-stranded DNA in bacteriophage capsids is both highly knotted and neatly structured. What is the physical basis of this organization? Here we show evidence from stochastic simulation techniques that suggests that a key element is the tendency of contacting DNA strands to order, as in cholesteric liquid crystals. This interaction favors their preferential juxtaposition at a small twist angle, thus promoting an approximately nematic (and apolar) local order. The ordering effect dramatically impacts the geometry and topology of DNA inside phages. Accounting for this local potential allows us to reproduce the main experimental data on DNA organization in phages, including the cryo-EM observations and detailed features of the spectrum of DNA knots formed inside viral capsids. The DNA knots we observe are strongly delocalized and, intriguingly, this is shown not to interfere with genome ejection out of the phage.