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Les urgentistes sont fréquemment confrontés à des décisions de limitation ou arrêt des thérapeutiques (LAT) en fin de vie. Il revient à l’urgentiste à la fois de décider la LAT et d’organiser les conditions de la fin de vie. Appliqué à des situations toujours singulières, parfois dramatiques, et dans lesquelles manquent souvent des informations à la phase initiale, l’exercice peut se révéler difficile. Sur le plan social et médiatique, la question de la fin de vie est récurrente. En seulement 20 ans, trois lois sont venues encadrer les décisions de LAT. Si la majorité de ces affaires ne concernaient pas directement les urgences, elles finissent par les impacter, car le problème de la fin de vie est universel. De plus, les urgentistes sont obligés d’appliquer les lois concernant la fin de vie, quand bien même celles-ci ne reconnaissent aucune spécificité à la mise en oeuvre des LAT en urgence. Que ce soit dans l’espace médiatique, professionnel ou privé, la parole d’un médecin urgentiste sur ce sujet sensible doit reposer sur une bonne connaissance des faits. Il en va de la crédibilité de la discipline en particulier et de la médecine en général. Il nous paraît donc intéressant de présenter une rétrospective de l’évolution législative ou réglementaire au sujet de la fin de vie en France depuis les années 1970, en montrant comment cette évolution a été intriquée avec les grandes affaires médiatisées survenues à la même époque.

International audience; Bio-based star-shaped poly(ε-caprolactone)s (S-PCL) derived from sugar-based D-sorbitol as an initiator were obtained via solvent-free enzymatic ring-opening polymerization (eROP). The star S-PCL were converted into UV-curable maleates by employing maleic anhydride for subsequent crosslinking with tri(ethylene glycol) divinyl ether (DVE-3) in the presence of Darocur 1173 as a radical photoinitiator. The kinetics of the UV-induced radical copolymerization was monitored by real-time Fourier-Transform InfraRed (FTIR) spectroscopy, which revealed that the star S-PCL maleate/divinyl ether system was not scavenged by molecular oxygen (donor/acceptor polymerization). The UV-crosslinking reaction was fast (~10 s) to reach near quantitative conversions. The S-PCL maleate / divinyl ether liquid formulation cast on glass substrates successfully gave films upon UV-crosslinking. The thermal properties of the polymer films and their precursor polymers were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Finally, the crosslinked polymer film demonstrated promising adhesive properties on steel, aluminum and glass substrates.

Pediatric central nervous system tumors are one of the most frequent types of neoplasms in children but epidemiological data on these tumors have been sparsely reported in the medical literature. We analyze the epidemiology of this type of tumors performing a retrospective population-based study in pediatrics and adolescent age in the population of Girona and compare them with series from Spain, Europe and worldwide. Cases were registered using the International Classification of Disease for Oncology, third edition and grouping according the International Classification of Childhood Cancer, third edition (ICCC-3). For all the histologies and the whole population between 0 and 19 years old, ASRw was 41.8 cases per million person-years. In children population, meaning under 14 years old, we found 104 cases with ASRw of 45.6. Males were the most affected by CNS tumors with a 1.2 sex ratio between 0 and 14 years old, and 1.1 between 0 and 19 years old. The analysis of trends in incidence did not find any statistically significant increase or decrease. Five-year observed survival was 68%, both for patients under 19 and 14 years of age. The incidence in our area was among the highest in Spain and worldwide, while survival was comparable to others reported.

To use the theoretical frameworks of implementation science to implement pressure injury (PI) prevention best practices in spinal cord injury (SCI) rehabilitation.Quality improvement.Six Canadian SCI rehabilitation centers.Inpatients (N=2371) admitted from 2011 to 2015.The SCI Knowledge Mobilization Network (SCI KMN) selected and implemented 2 PI prevention best practices at 6 Canadian SCI rehabilitation centers: (1) completing a comprehensive PI risk assessment comprised of a structured risk assessment instrument followed by an individualized, interprofessional risk factor determination and prevention plan; and (2) providing structured and individualized PI prevention patient education. Active Implementation Frameworks provided a systematic approach to best practice implementation.Implementation indicators (completion rates) and patient outcomes (PI incidence, patient education survey).After implementation, risk assessment completion rates improved from 46% to 94% (P.05). Between initial (2012-2013) and full (2014-2015) implementation stages, completion rates improved for both interprofessional risk factor determination (67% to 96%) and prevention plans (67% to 94%). Documentation of patient education also increased to 86% (vs. 71% preimplementation). At rehabilitation admission 22% of patients had PIs, with 14% of individuals developing new PIs during rehabilitation. The overall PI prevalence was 30%. Considering only PIs of stage 2 or greater, prevalence was 21% and incidence 7%. There were no statistically significant differences in PI incidence between pre- and postimplementation. Patient education surveys indicated that PI education improved patients' knowledge of prevention strategies.Active Implementation Frameworks supported successful implementation of PI prevention best practices across the 6 participating SCI KMN sites. Achieving a reduction in PI incidence will require additional measures, and there is an ongoing need to strengthen the evidence base underpinning PI prevention guidelines.

Cell line development (CLD) by random integration (RI) can be labor intensive, inconsistent, and unpredictable due to uncontrolled gene integration after transfection. Unlike RI, targeted integration (TI) based CLD introduces the antibody-expressing cassette to a predetermined site by recombinase-mediated cassette exchange (RMCE). The key to success for the development of a TI host for therapeutic antibody production is to identify a transcriptionally active hotspot that enables highly efficient RMCE and antibody expression with good stability. In this study, a genome wide search for hotspots in the Chinese hamster ovary (CHO)-K1-M genome by either RI or PiggyBac (PB) transposase-based integration has been described. Two CHO-K1-M derived TI host cells were established with the Cre/Lox RMCE system and are described here. Both TI hosts contain a GFP-expressing landing pad flanked by two incompatible LoxP recombination sites (L3 and 2L). In addition, a third incompatible LoxP site (LoxFAS) is inserted in the GFP landing pad to enable an innovative two-plasmid based RMCE strategy, in which two separate vectors can be targeted to a single locus simultaneously. Cell lines generated by the TI system exhibit comparable or higher productivity, better stability and fewer sequence variant (SV) occurrences than the RI cell lines.

Resume Cet article etudie les representations que les professionnels du monde judiciaire se font des personnes placees sous main de justice (PPSMJ) dont ils ont la charge. Une comparaison selon les institutions concernees est pertinente dans la mesure ou les missions des policiers, des psychiatres, des CPIP et des travailleurs sociaux different et donc leur relation avec les PPSMJ n’est pas de meme nature. Une enquete de terrain realisee a l’aide d’entretiens semi-directifs qui ont fait l’objet d’analyses de discours et de contenu permet de rendre compte des particularites liees aux interactions sociales menees par ces differents professionnels. Schematiquement, les policiers manifestent une mise a distance des PPSMJ, les psychiatres, respectueux du secret medical sont centres sur la PPSMJ en tant que source de son traitement, les CPIP hesitent entre leur role social et leur role repressif et les travailleurs sociaux, tres investis dans la relation avec les PPSMJ dont ils ont la charge, evacuent les causes de leur desocialisation. Les representations que ces differents professionnels se font des PPSMJ font apparaitre la necessite de comparer ces representations avec les attentes des PPSMJ dont ils s’occupent de maniere a proposer des preconisations pour reduire les ecarts et ameliorer ainsi la relation entre les differents protagonistes de l’interaction.

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)-enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND-enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4 months at Genentech), leading to a potential acceleration of 4 months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies-mAb1, mAb2, and mAb3-at the 2 L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2 L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400 L scale and three GMP batches at 2000 L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND-enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1449-1455, 2017.

Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

Recently, several health authorities have requested substantial detail from sponsor firms regarding the practices employed to generate the production cell line for recombinant DNA-(rDNA) derived biopharmaceuticals. Two possible inferences from these regulatory agency questions are that (1) assurance of "clonality" of the production cell line is of major importance to assessing the safety and efficacy of the product and (2), without adequate proof of "clonality", additional studies of the cell line and product are often required to further ensure the product's purity and homogeneity. Here we address the topic of "clonality" in the broader context of product quality assurance by current technologies and practices, as well as discuss some of the relevant science and historical perspective. We agree that the clonal derivation of a production cell line is one factor with potential impact, but it is only one of many factors. Further, we believe that regulatory emphasis should be primarily placed on ensuring product quality of the material actually administered to patients, and on ensuring process consistency and implementing appropriate control strategies through the life cycle of the products.

Objectif Evaluer l’association entre l’incidence d’une dysthyroidie induite et l’efficacite therapeutique d’une immunotherapie anti-checkpoints par anti-PD-1 (Nivolumab) dans une cohorte de patients traites pour un cancer du poumon non a petites cellules (CPNPC). Materiels et methodes Dans cette etude de cohorte retrospective etaient inclus les patients atteints d’un CPNPC de stade IIIB/IV confirme histologiquement, progressif et ayant initie un traitement par Nivolumab. La fonction thyroidienne (TSH ± T4L, T3L) etait surveillee et les patients etaient classes en fonction de l’incidence d’une dysthyroidie (Dysthyroidie (+) et Dysthyroidie (-)) et selon la severite et le sous-type de dysthyroidie. Les criteres principaux de jugement etaient la survie globale (SG) et la survie sans progression (SSP). Resultats Cent trente-quatre patients (âge median : 63 ans ; 70,1 % d’hommes) etaient inclus. Quarante patients (29,9 %) ont ete classes dans le groupe Dysthyroidie (+) et avaient une SG mediane plus longue de 29,8 mois (15,2-non atteinte) contre 8,1 mois (3-20,4) dans le groupe Dysthyroidie (-) (p Conclusion Notre etude suggere que la dysthyroidie induite par Nivolumab est un facteur predictif d’efficacite therapeutique chez les patients traites pour un CPNPC, independamment de la severite ou du sous-type de dysthyroidie.

The source-to-sink (S2S) concept focuses on the quantification of the components of clastic sedimentary systems from initial sediment production in the source areas (i.e. onshore margin) through to their deposition within sedimentary sinks (i.e. offshore margin, Somme et al. 2016, Bhattacharya et al. 2016). Clastics provenance analysis provides fundamental information about source and catchment areas as well as the sedimentary budget marking the offshore margin domain. Clastics production on the margin is governed by the mechanical erosion and/or the chemical weathering of the onshore substrate. The intensity of the onshore erosion processes is mainly controlled by the lithology of the substrate, the rate of tectonic uplift, and the type of paleoclimate (hot humid climate favoring weathering versus cold dry climate favoring mechanical erosion). The main objective of the South Angola margin S2S project is to characterize (1) the factors controlling the intensity of the onshore erosional processes and (2) the volume and nature of the Cretaceous to Cenozoic clastics produced in the South Angola onshore domains and delivered to the offshore margin.

In the development of biopharmaceutical products, the expectation of regulatory agencies is that the recombinant proteins are produced from a cell line derived from a single progenitor cell. A single limiting dilution step followed by direct imaging, as supplemental information, provides direct evidence that a cell line originated from a single progenitor cell. To obtain this evidence, a high-throughput automated imaging system was developed and characterized to consistently ensure that cell lines used for therapeutic protein production are clonally-derived. Fluorescent cell mixing studies determined that the automated imaging workflow and analysis provide ∼95% confidence in accurately and precisely identifying one cell in a well. Manual inspection of the images increases the confidence that the cell line was derived from a single-cell to >99.9%. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:584-592, 2018.

International audience; Cryomodules of the superconducting accelerator SPIRAL 2 have been successfully qualified and are now under commissioning on the linac at GANIL (France). This paper presents the successful results of the power conditioning of the couplers both on a test bench in Grenoble and during the cryomodules qualification. It also shows the influence of some factors, such as surface state and experiencing a cavity quench around the antenna, on the power conditioning process (duration, quality).

Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3.

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR-deficient DG44, and DUXB11-based DHFR deficient CHO. Current Genentech commercial full-length antibody products have all been produced in the DUXB11-derived DHFR-deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11-derived DHFR-deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11-based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14-day fed batch cultures in shake flasks. In contrast, the DUXB11-based host produced ∼0.1 g/l for both antibodies in the same 14-day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ∼2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass.

The FP7 project PROGRESS aims at improving the security and resilience of European space systems such as GNSS (Global Navigation Satellite Systems). It focuses on the detection and mitigation of attacks on ground-based infrastructures from highly educated attackers whose numbers may increase in the near future. This paper outlines the main project objectives and presents the work achieved in this framework considering the High Power Microwaves (HPM) threats. Development and characterization of a HPM detector are reported as well as the work carried out in terms of infrastructure protection.

Resume Malgre des avantages reconnus aux antipsychotiques par voie injectable, il existe encore des reticences face a ces traitements, tant de la part de certains patients que de la part des professionnels de sante. Une enquete realisee en 2007 aupres de patients souffrant de trouble schizophrenique avait montre que pres de la moitie de ces patients preferait une forme injectable et se sentaient ainsi mieux accompagnes. Il existe aujourd’hui la possibilite de choisir entre deux sites d’injection : le muscle deltoide ou le muscle gluteal. L’enquete presentee ici (realisee par l’Institut de sondage BVA en 2011) vise a recueillir le point de vue des patients sur ce nouveau choix et les changements qu’il induit. Deux cent quatre-vingts un patients avec une maladie schizophrenique suivis en ambulatoire et sous traitement injectable a action prolongee ont ete interroges. Il ressort de cette enquete que 75 % des patients disent se sentir mieux suite a la prise d’un traitement injectable. Le choix du site d’injection, montrant une preference pour le muscle deltoide, est important pour une majorite (70 %), renforcant leur sentiment d’etre acteur de leur traitement. Ils se sentent ainsi plus impliques dans le suivi de celui-ci, ce qui devrait favoriser une meilleure observance.

Resume L’interdisciplinarite n’est pas donnee d’avance. Elle se construit au jour le jour. Elle est cependant une exigence de plus en plus necessaire a la prise en charge des patients aujourd’hui, particulierement en fin de vie. Nous essaierons de definir ce qu’est l’interdisciplinarite et de repondre aux questions suivantes : Quels en sont les enjeux ? Comment se met-elle en pratique et quelles en sont les conditions ? Quels en sont les ecueils, en termes de vie de groupe (en particulier l’illusion groupale) et de decision commune ? Quels champs recoupe-elle ? Se limite-t-elle a la prise en charge clinique des patients ? Quelles ouvertures apporte-t-elle et en quoi est-elle fondamentale ?

Protein glycation is a non-enzymatic glycosylation that can occur to proteins in the human body, and it is implicated in the pathogenesis of multiple chronic diseases. Glycation can also occur to recombinant antibodies during cell culture, which generates structural heterogeneity in the product. In a previous study, we discovered unusually high levels of glycation (>50%) in a recombinant monoclonal antibody (rhuMAb) produced by CHO cells. Prior to that discovery, we had not encountered such high levels of glycation in other in-house therapeutic antibodies. Our goal here is to develop cell culture strategies to decrease rhuMAb glycation in a reliable, reproducible, and scalable manner. Because glycation is a post-translational chemical reaction between a reducing sugar and a protein amine group, we hypothesized that lowering the concentration of glucose--the only source of reducing sugar in our fed-batch cultures--would lower the extent of rhuMAb glycation. When we decreased the supply of glucose to bioreactors from bolus nutrient and glucose feeds, rhuMAb glycation decreased to below 20% at both 2-L and 400-L scales. When we maintained glucose concentrations at lower levels in bioreactors with continuous feeds, we could further decrease rhuMAb glycation levels to below 10%. These results show that we can control glycation of secreted proteins by controlling the glucose concentration in the cell culture. In addition, our data suggest that rhuMAb glycation occurring during the cell culture process may be approximated as a second-order chemical reaction that is first order with respect to both glucose and non-glycated rhuMAb. The basic principles of this glycation model should apply to other recombinant proteins secreted during cell culture.

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.

An amino acid sequence variant is defined as an unintended amino acid sequence change and contributes to product heterogeneity. Recombinant monoclonal antibodies (MAbs) are primarily expressed from Chinese Hamster Ovary (CHO) cells using stably transfected production cell lines. Selections and amplifications with reagents such as methotrexate (MTX) are often required to achieve high producing stable cell lines. Since MTX is often used to generate high producing cell lines, we investigated the genomic mutation rates of the hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT) gene using a 6-thioguanine (6-TG) assay under various concentrations of MTX selection in CHO cells. Our results show that the 6-TG resistance increased as the MTX concentration increased during stable cell line development. We also investigated low levels of sequence variants observed in two stable cell lines expressing different MAbs. Our data show that the replacement of serine at position 167 by arginine (S167R) in the light chain of antibody A (MAb-A) was due to a genomic nucleotide sequence change whereas the replacement of serine at position 63 by asparagine (S63N) in the heavy chain of antibody B (MAb-B) was likely due to translational misincorporation. This mistranslation is codon specific since S63N mistranslation is not detectable when the S63 AGC codon is changed to a TCC or TCT codon. Our results demonstrate that both a genomic nucleotide change and translational misincorporation can lead to low levels of sequence variants and mistranslation of serine to asparagine can be eliminated by substituting the TCC or TCT codon for the S63 AGC codon without impacting antibody productivity.

The direct sea–land correlation applied to core MD99-2331 retrieved from the north-western Iberian margin shows a two-phase pattern within Heinrich events 4, 2 and 1 in the ocean and in the adjacent landmasses. Changes between wet/cold and dry/cool conditions in the Iberian Peninsula detected during these extreme events cannot be explained by a simple oceanographic mechanism related to changes in the strength of the Atlantic Meridional Overturning Circulation. Here we propose an additional atmospheric mechanism able to produce this scenario based on the comparison between the MD99-2331 record and other available palaeoclimate sequences from the North Atlantic region (18–75°N and 0–75°W). The climatic asymmetry observed between mid- and subtropical eastern North Atlantic latitudes (wet/dry) and the Blake Outer Ridge (dry/wet) during H4, H2 and H1 can be explained by changes in the position of the Atlantic jet-stream. During the first phase of H4, H2 and H1 the Atlantic jet-stream was located further south following the southward displacement of the oceanic thermal front as far south as 35°–37°N. On the contrary, during the second phase of H4, H2 and H1 the jet-stream was located further north following the northward displacement of this thermal front as far north as 42°N. From the atmospheric point of view, these two phases are reminiscent of the present-day negative and positive prevailing modes of the North Atlantic Oscillation (NAO), respectively, but high-resolution studies of additional North Atlantic key sites and climate simulations are needed to confirm the hypothesis of a NAO-like mechanism operating on millennial timescales.

Les angoisses de nos concitoyens concernant les conditions de leur fin de vie sont de plus en plus grandes et notre medecine tres performante et technique est parfois vecue comme deshumanisee. Les professionnels de sante doivent exercer parfois dans des situations extremement complexes comme dans le cas ou des decisions de limitations ou d’arret de traitement doivent etre prises. Dans le souci d’ameliorer les conditions de la fin de vie, la loi n° 2005-370 du 22 avril 2005 relative aux droits des malades et a la fin de vie introduit pour la premiere fois en France les directives anticipees aux cotes de la procedure collegiale et de la transparence des pratiques. Les directives anticipees donnent la parole au patient et lui permettent de s’impliquer dans son projet de soins. En encourageant le dialogue sur la fin de vie, elles favorisent l’etablissement d’une relation de confiance medecin – malade. Les directives anticipees peuvent guider les professionnels de soin dans les decisions necessitant une evaluation globale des interets du patient et les sensibilisent au besoin d’une prise en charge toujours plus singuliere et humaine. Elles nous renvoient aux notions d’autonomie et de competence, de paternalisme et de bienfaisance medicale. A la lumiere de ces questionnements et de notre experience, nous proposons ici des recommandations pratiques d’elaboration et d’utilisation des directives anticipees.

Central venous catheter (CVC)-related infections (CRIs) are a key target for infection control in intensive care units (ICUs). The aim of this study was to describe temporal trends of CRI incidence in a network of volunteer ICUs in Northern France. During a 4 month surveillance period each year, all CVCs in place for more than 48h were prospectively followed until removal or patient discharge. Standard clinical and microbiological criteria were used to define colonization and CRI. The standardized incidence ratio (SIR) was estimated by dividing the number of observed CRIs by the number of expected CRIs, which was computed using a logistic regression model including risk factors for CRI. CRI incidence and SIR were fed back to ICUs as a benchmark at the end of each period. From 2001 to 2005, 135 ICUs participated for at least one surveillance period. Overall, 11 703 CVC in 9182 patients (122 495 CVC-days) were included. CRI incidence was 2.8 per 1000 CVC-days. Among 35 ICUs that participated for three or more consecutive periods, CRI incidence decreased significantly by 58.6%. SIR also decreased significantly from the first to the third surveillance period in these ICUs. These results suggest that surveillance programmes have a significant impact on CRI risk in ICUs and remain an important strategy for combating nosocomial infections in these settings.

Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

Resume Objectif Le but de l’etude est d’objectiver l’influence de la nouvelle version de la norme de mesurage du bruit NF S 31-084 sur le niveau sonore releve par dosimetrie individuelle dans le domaine de la maintenance aeronautique. Methode Une centaine de dosimetries individuelles de bruit de mecaniciens aeronautiques ont ete traitees selon la nouvelle version de la norme NF S 31-084. Les valeurs d’exposition au bruit calculees selon la norme dans differents groupes d’exposition homogenes (GEH) ont ete comparees aux valeurs moyennes d’exposition anterieurement utilisees. Resultats Les valeurs d’exposition au bruit calculees selon la nouvelle version de la norme sont significativement plus elevees (4 a 10 dBA) que les valeurs moyennes de chaque groupe d’exposition. Les niveaux sonores des mecaniciens aeronautiques selon la nouvelle version de la norme sont de l’ordre de 88 dB(A) pour les activites au hangar, et de l’ordre de 92 dB(A) pour le travail en piste. Conclusion En integrant l’incertitude de mesure et l’incertitude d’echantillonnage dans le but de garantir une meilleure representativite des mesures de bruit, la nouvelle version de la norme NF S 31-084 donne des evaluations considerablement plus elevees de l’exposition au bruit dans un milieu ou le niveau sonore est tres fluctuant par nature. Si ce mode de calcul est plus protecteur au sens reglementaire pour les salaries, il ne traduit pas pour autant une aggravation de leurs conditions de travail.

Position du probleme La surveillance epidemiologique des infections nosocomiales est l’un des axes importants de l’amelioration de la qualite des soins dans les etablissements de sante. Les services participant a un reseau ayant des methodes standardisees peuvent etre compares entre eux en utilisant des indices de risque ajustes. Au sein du reseau de surveillance des infections liees aux catheters veineux centraux (ILC) en reanimation (REACAT), nous proposons un indice de risque ajuste sur plusieurs facteurs de risque des ILC : le ratio standardise d’incidence (RSI). Methodes Tous les catheters veineux centraux (CVC) maintenus plus de 48h etaient surveilles en prospectif, 4 mois par an. Les ILC etaient definies sur des criteres cliniques et microbiologiques consensuels. Un modele de regression logistique, developpe sur les donnees regroupees des trois dernieres periodes de surveillance, a ete retenu comme modele predictif de l’ILC. Pour chaque service et annee, un nombre d’ILC attendu a ete calcule et a permis d’estimer le RSI. Resultats Entre 2000 et 2003, 108 services ont participe a au moins une des periodes de surveillance, incluant 6 414 CVC. Deux cent trente neuf ILC ont ete identifiees, soit une densite d’incidence (DI) de 3,6 ILC/1000 jours d’exposition aux CVC. Les facteurs associes a l’ILC etaient la duree de maintien (OR ajuste = 1,1 ; IC95 % [1,0-1,1]), le rang de pose (1,7 ; [1,1-2,2]), le site d’insertion (1,6 ; [1,2-2,1]), la perfusion d’antibiotique (0,5 [0,3-0,7]), l’existence d’au moins une defaillance viscerale (2,2 [1,5-3,2] ) et d’une infection a un autre site a l’ablation du CVC (1,9 [1,4-2,6]). Parmi les 14 services avec une DI superieure a 5,5 ILC/1 000 jours-CVC (correspondant au 75 e percentile), seulement 2 avaient un RSI significativement superieur a 1, dont un seul avait inclus plus de 20 CVC. Conclusion La surveillance REACAT a permis la construction d’un RSI, indice de risque global et robuste, permettant d’identifier des services ou le risque d’ILC est eleve et d’orienter les actions de prevention.

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t -test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield.

The aim of this study was to evaluate the number of deaths associated with nosocomial infections (NI) and the contribution of these NI to death. A multicentre descriptive study was conducted in 16 tertiary-care hospitals (14 222 beds) in Northern France. Medical records of consecutive patients who died at least 48 h after admission were reviewed for cause of death, NI and disease severity, before admission and before NI onset. The contribution of NI to death was assessed by agreement between two physicians according to a three-category scale of probability. Among the 1945 patients who died during the study, 26.6% had an NI. According to the agreed diagnosis, NI contributed to the deaths of 284 (14.6%) patients (certainly for 6.6% and possibly for 8%), thereby ranking NI as the fourth most frequent cause of death. Considering the deaths that had not been anticipated independently of NI two weeks before they occurred, NI definitely contributed to 2.8% of them. Lower respiratory tract, bloodstream and surgical wound infections were responsible for 39, 20 and 14%, respectively, of all NI in these patients. The impact of NI on in-hospital mortality seems to be lower than had previously been estimated in France based on US data from the 1970s and 1980s. To improve healthcare quality, further studies are needed to elucidate the processes that may contribute to fatal severe NI.

Une grossesse apres chimiotherapie d’un cancer du sein n’est plus un evenement rare. L’exemple de ces deux femmes ayant debute une grossesse spontanee apres chimiotherapie en est la preuve. L’information des patientes, la connaissance des medicaments et de leurs effets secondaires ainsi que les possibilites de procreation medicalement assistee permettent aux femmes qui le souhaitent d’envisager une grossesse dans des conditions de securite pour la mere et l’enfant.

Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines. In the first cell line, when extracellular copper remained above the limit of detection (LOD), cultures shifted to net lactate consumption and yielded comparable performances irrespective of the differences in copper levels; when extracellular copper dropped below LOD (∼13 nM), cultures failed to shift to net lactate consumption, and yielded significantly lower product titers. Across the four cell lines, the ability to grow and consume lactate seemed to depend on the presence of a minimum level of copper, beyond which there were no further gains in culture performance. Although this minimum cellular copper requirement could not be directly quantified, we estimated its probable range for the first cell line by applying several assumptions. Even when different copper concentrations did not affect cell culture performance, they affected product quality profiles: higher initial copper concentrations increased the basic variants in the recombinant IgG1 products. Therefore, in optimizing chemically defined media, it is important to select a copper concentration that is adequate and achieves desired product quality attributes.

Reducing dietary phosphorus (P) is a common approach to reduce effluent P outputs. The potential resulting P-deficiency is known to negatively impact fish bone condition and might result in vertebral deformities. To date, no large-scale study involving deep sequencing of the bone transcriptome has been conducted in salmonids and vertebral molecular changes remain poorly described. This study aims to provide the first comprehensive vertebral transcriptome for rainbow trout (Oncorhynchus mykiss) to allow functional and quantitative expression studies. Fish weighing 60.8±1.6g, were fed for 27weeks using two practical diets having 0.29% (deficient) and 0.45% (sufficient) available phosphorus (P), respectively. Deep sequencing was conducted using HiSeq2000 Illumina 100 paired-end technology from pooled P-deficient and P-sufficient fish and individuals displaying vertebral deformities. Over 140 million trimmed paired-end reads were assembled de novo with Trinity and resulted in 679,869 transcripts with a mean length of 542.5bp. From these sequences, 340,747 matched with referenced ESTs from rainbow trout. Furthermore, 141,909 and 117,564 sequences were functionally annotated against Nr and Uniprot databases, respectively. Interestingly, we observed putative homologue sequences for most of the key components involved in bone formation and turnover in mammals.

The biotechnology industry has exploited the unique properties of the periplasm to produce industrially relevant products. For efficient periplasmic protein accumulation to occur, events in the cytoplasm related to translation initiation and protein folding of the mature domain need to be coordinated. In this chapter the authors demonstrate that nucleotide changes in the translation initiation region of the gene profoundly influenced periplasmic protein accumulation. There are many documented examples of cytoplasmic proteins having positive effects on periplasmic protein accumulation of endogenous or recombinant proteins. A possible molecular explanation is that the cytoplasmic resident protein is preventing the preprotein from negative pathways, such as premature folding and inclusion body formation, and keeping the protein in an unfolded state and allowing efficient export. There are three possible fates for newly secreted proteins upon release into the periplasmic space. First, proteins can misfold and aggregate forming insoluble inclusion bodies. Second, proteins can be proteolyzed by endogenous periplasmic and membrane proteases where the protease domain of membrane proteases is located in the periplasm. Lastly, proteins can successfully fold into the correct conformation and exist as soluble, biologically active proteins. The lack of an identified chaperone system in the periplasm has been hindering the advancement of recombinant periplasmic protein accumulation. The combination of over-expression of periplasmic components participating in protein-folding pathways and the inclusion of chemical additives has been reported and shown to have positive benefits.