How family caregivers' medical and moral assumptions influence decision making for patients in the vegetative state: a qualitative interview study.

Background Decisions on limiting life-sustaining treatment for patients in the vegetative state (VS) are emotionally and morally challenging. In Germany, doctors have to discuss, together with the legal surrogate (often a family member), whether the proposed treatment is in accordance with the patient's will. However, it is unknown whether family members of the patient in the VS actually base their decisions on the patient's wishes. Objective To examine the role of advance directives, orally expressed wishes, or the presumed will of patients in a VS for family caregivers' decisions on life-sustaining treatment. Methods and sample A qualitative interview study with 14 next of kin of patients in a VS in a long-term care setting was conducted; 13 participants were the patient's legal surrogates. Interviews were analysed according to qualitative content analysis. Results The majority of family caregivers said that they were aware of aforementioned wishes of the patient that could be applied to the VS condition, but did not base their decisions primarily on these wishes. They gave three reasons for this: (a) the expectation of clinical improvement, (b) the caregivers' definition of life-sustaining treatments and (c) the moral obligation not to harm the patient. If the patient's wishes were not known or not revealed, the caregivers interpreted a will to live into the patient's survival and non-verbal behaviour. Conclusions Whether or not prior treatment wishes of patients in a VS are respected depends on their applicability, and also on the medical assumptions and moral attitudes of the surrogates. We recommend repeated communication, support for the caregivers and advance care planning.

Nonself vegetative fusion and genetic exchange in the arbuscular mycorrhizal fungus Glomus intraradices.

Arbuscular mycorrhizal fungi (AMF) form symbioses with the majority of plants and form extensive underground hyphal networks simultaneously connecting the roots of different plant species. No empirical evidence exists for either anastomosis between genetically different AMF or genetic exchange.Five isolates of one population of Glomus intraradices were used to study anastomosis between hyphae of germinating spores. We show that genetically distinct AMF, from the same field, anastomose, resulting in viable cytoplasmic connections through which genetic exchange could potentially occur.Pairs of genetically different isolates were then co-cultured in an in vitro system.Freshly produced spores were individually germinated to establish new cultures.Using several molecular tools, we show that genetic exchange occurred between genetically different AMF. Specific genetic markers from each parent were transmitted to the progeny. The progeny were viable, forming symbioses with plant roots. The phenotypes of some of the progeny were significantly different from either parent.Our results indicate that considerable promiscuity could occur in these fungi because nine out of 10 combinations of different isolates anastomosed. The ability to perform genetic crosses between AMF experimentally lays a foundation for understanding the genetics and evolutionary biology of these important plants symbionts.

Sexual dimorphism in resource acquisition and deployment: both size and timing matter.

Background and Aims The males and females of many dioecious plant species differ from one another in important life-history traits, such as their size. If male and female reproductive functions draw on different resources, for example, one should expect males and females to display different allocation strategies as they grow. Importantly, these strategies may differ not only between the two sexes, but also between plants of different age and therefore size. Results are presented from an experiment that asks whether males and females of Mercurialis annua, an annual plant with indeterminate growth, differ over time in their allocation of two potentially limiting resources (carbon and nitrogen) to vegetative (below-and above-ground) and reproductive tissues.Methods Comparisons were made of the temporal patterns of biomass allocation to shoots, roots and reproduction and the nitrogen content in the leaves between the sexes of M. annua by harvesting plants of each sex after growth over different periods of time.Key Results and Conclusions Males and females differed in their temporal patterns of allocation. Males allocated more to reproduction than females at early stages, but this trend was reversed at later stages. Importantly, males allocated proportionally more of their biomass towards roots at later stages, but the roots of females were larger in absolute terms. The study points to the important role played by both the timing of resource deployment and the relative versus absolute sizes of the sinks and sources in sexual dimorphism of an annual plant.

Prognostication of neurologic outcome in cardiac arrest patients after mild therapeutic hypothermia: a meta-analysis of the current literature.

PURPOSE: To assess the sensitivity and false positive rate (FPR) of neurological examination and somatosensory evoked potentials (SSEPs) to predict poor outcome in adult patients treated with therapeutic hypothermia after cardiopulmonary resuscitation (CPR). METHODS: MEDLINE and EMBASE were searched for cohort studies describing the association of clinical neurological examination or SSEPs after return of spontaneous circulation with neurological outcome. Poor outcome was defined as severe disability, vegetative state and death. Sensitivity and FPR were determined. RESULTS: A total of 1,153 patients from ten studies were included. The FPR of a bilaterally absent cortical N20 response of the SSEP could be calculated from nine studies including 492 patients. The SSEP had an FPR of 0.007 (confidence interval, CI, 0.001-0.047) to predict poor outcome. The Glasgow coma score (GCS) motor response was assessed in 811 patients from nine studies. A GCS motor score of 1-2 at 72 h had a high FPR of 0.21 (CI 0.08-0.43). Corneal reflex and pupillary reactivity at 72 h after the arrest were available in 429 and 566 patients, respectively. Bilaterally absent corneal reflexes had an FPR of 0.02 (CI 0.002-0.13). Bilaterally absent pupillary reflexes had an FPR of 0.004 (CI 0.001-0.03). CONCLUSIONS: At 72 h after the arrest the motor response to painful stimuli and the corneal reflexes are not a reliable tool for the early prediction of poor outcome in patients treated with hypothermia. The reliability of the pupillary response to light and the SSEP is comparable to that in patients not treated with hypothermia.

Reversible behavioural autistic-like regression: A manifestation of a special (new?) epileptic syndrome in a 28-month-old child. A 2-year longitudinal study .

A 28-month-old boy was referred for acute onset of abnormal head movements. History revealed an insidious progressive regression in behaviour and communication over several months. Head and shoulder 'spasms' with alteration of consciousness and on one occasion ictal laughter were seen. The electroencephalograph (EEG) showed repeated bursts of brief generalized polyspikes and spike-wave during the 'spasms', followed by flattening, a special pattern which never recurred after treatment. Review of family videos showed a single 'minor' identical seizure 6 months previously. Magnetic resonance imaging was normal. Clonazepam brought immediate cessation of seizures, normalization of the EEG and a parallel spectacular improvement in communication, mood and language. Follow-up over the next 10 months showed a new regression unaccompained by recognized seizures, although numerous seizures were discovered during the videotaped neuropsychological examination, when stereotyped subtle brief paroxysmal changes in posture and behaviour could be studied in slow motion and compared with the 'prototypical' initial ones. The EEG showed predominant rare left-sided fronto-temporal discharges. Clonazepam was changed to carbamazepin with marked improvement in behaviour, language and cognition which has been sustained up to the last control at 51 months. Videotaped home observations allowed the documentation of striking qualitative and quantitative variations in social interaction and play of autistic type in relation to the epileptic activity. We conclude that this child has a special characteristic epileptic syndrome with subtle motor and vegetative symptomatology associated with an insidious catastrophic 'autistic-like' regression which could be overlooked. The methods used to document such fluctuating epileptic behavioural manifestations are discussed.

Prognostication after cardiac arrest and hypothermia: a prospective study.

Current American Academy of Neurology (AAN) guidelines for outcome prediction in comatose survivors of cardiac arrest (CA) have been validated before the therapeutic hypothermia era (TH). We undertook this study to verify the prognostic value of clinical and electrophysiological variables in the TH setting. A total of 111 consecutive comatose survivors of CA treated with TH were prospectively studied over a 3-year period. Neurological examination, electroencephalography (EEG), and somatosensory evoked potentials (SSEP) were performed immediately after TH, at normothermia and off sedation. Neurological recovery was assessed at 3 to 6 months, using Cerebral Performance Categories (CPC). Three clinical variables, assessed within 72 hours after CA, showed higher false-positive mortality predictions as compared with the AAN guidelines: incomplete brainstem reflexes recovery (4% vs 0%), myoclonus (7% vs 0%), and absent motor response to pain (24% vs 0%). Furthermore, unreactive EEG background was incompatible with good long-term neurological recovery (CPC 1-2) and strongly associated with in-hospital mortality (adjusted odds ratio for death, 15.4; 95% confidence interval, 3.3-71.9). The presence of at least 2 independent predictors out of 4 (incomplete brainstem reflexes, myoclonus, unreactive EEG, and absent cortical SSEP) accurately predicted poor long-term neurological recovery (positive predictive value = 1.00); EEG reactivity significantly improved the prognostication. Our data show that TH may modify outcome prediction after CA, implying that some clinical features should be interpreted with more caution in this setting as compared with the AAN guidelines. EEG background reactivity is useful in determining the prognosis after CA treated with TH.

Role of penicillin-binding proteins in Streptococcus gordonii

Résumé Streptococcus gordonii est une bactérie colonisatrice naturelle de la cavité buccale de l'homme. Bien que normalement commensale, elle peut causer des infections graves, telles que des bactériémies ou des endocardites infectieuses. La pénicilline étant un des traitements privilégiés dans de tels cas, l'augmentation rapide et globale des résistances à cet antibiotique devient inquiétante. L'étude de la physiologie et des bases génétiques de ces résistances chez S. gordonii s'avère donc importante. Les cibles moléculaires privilégiées de la pénicilline G et des β-lactames sont les penicilllin-binding proteins (PBPs). Ces enzymes associées à la membrane ont pour rôle de catalyser les réactions de transpeptidation et de transglycosylation, qui constituent les dernières étapes de la biosynthèse du peptidoglycan (PG). Elles sont définies comme classe A ou B selon leur capacité d'assurer soit les deux réactions, soit uniquement la transpeptidation. Les β-lactames inhibent le domaine transpeptidase de toutes les PBPs, entraînant l'inhibition de la synthèse du PG, l'inhibition de la croissance, et finalement la mort cellulaire. Chez les streptocoques, les PBPs sont aussi les premiers déterminants de la résistance à la pénicilline. De plus, elles sont impliquées dans la morphologie bactérienne, en raison de leur rôle crucial dans la formation du PG. Le but de ce travail était de caractériser les PBPs de S. gordonii et d'étudier leurs fonctions dans la vie végétative de la bactérie ainsi que durant le développement de la résistance à la pénicilline. Premièrement, des mutants auxquels il manque une ou deux PBP(s) ont été construits. Leur étude - au niveau physiologique, biochimique et morphologique - a montré le caractère essentiel ou dispensable de chaque protéine, ainsi que certaines de leurs fonctions potentielles. Deuxièmement, des mutants résistants à la pénicilline ont été générés. Leur caractérisation a montré l'importance des mutations dans les PBPs ainsi que dans d'autres gènes encore inconnus, de même que le rôle crucial des PBPs de classe A dans le développement de la résistance à la pénicilline. Des expériences supplémentaires sur des isolats résistants ont aussi prouvé que la résistance a un coût en terme de fitness, coût que S. gordonii parvient à compenser par des mécanismes d'adaptation. Finalement, les promoteurs des gènes des PBPs ont été déterminés et leur expression a été étudiée grâce au gène de luciférase. Il a ainsi été montré que la résistance à la pénicilline entraîne non seulement des altérations au niveau des protéines, mais aussi au niveau de la régulation des gènes. De plus, la pénicilline génère directement des modifications dans l'expression de PBPs spécifiques. Summary Streptococcus gordonii is a normal inhabitant of the human oral cavity and a pioneer colonizer of teeth. Although usually considered as a commensal, this organism can cause life-threatening infections such as bacteraemia or endocarditis. Since penicillin is one of the preferential treatments for such pathologies, the rapid and general increase of antibiotic resistance in the overall population becomes an issue. Thus, studying the physiologic and genetic bases of such a resistance in S. gordonii is of interest. The primary molecular targets of penicillin G and other β-lactams are the so called penicillin-binding proteins (PBPs). These are membrane-associated proteins that catalyze the last steps in peptidoglycan (PG) biosynthesis, namely transpeptidation and transglycosylation. Depending on their capacity to catalyze either reactions or only transpeptidation, they are considered as class A or class B PBPs, respectively. β-lactam antibiotics inhibit the transpeptidase domain of both of these classes of enzymes, resulting in inhibition of PG assembly, inhibition of bacterial growth, and ultimately leading to cell death. In streptococci, PBPs are also the primary determinants of penicillin-resistance. Moreover, because of their crucial role in PG formation, they are implicated in fundamental aspects of cell morphology. The goal of this work was thus to characterize S. gordonii PBPs and to explore their functions in terms of vegetative life and penicillin-resistance development. First, single and double PBP-inactivated mutants were generated and their effect on the bacterial physiology, cell wall biochemistry and ultrastructural morphology was assessed. This demonstrated the essentiality or dispensability of each protein for bacterial life. Second, penicillin-resistant mutants were generated by cyclic exposure to increasing concentrations of the drug. Characterization of these mutants pointed out the importance of both PBP and non-PBP mutations, as well as the crucial role of the class A PBPs in the development of penicillin-resistance. Further experiments on resistant isolates demonstrated the fitness cost of this resistance, but also the capacity of S. gordonii to adapt and regain the fitness of the wild-type. Finally, the promoters of PBP genes were determined and their expression was monitored using luciferase fusions. This showed that penicillin-resistance, in addition to modifications at the level of the protein, also triggered genetic alterations. Moreover, penicillin itself generated modifications in the expression of specific PBPs.

Inducible malondialdehyde pools in zones of cell proliferation and developing tissues in Arabidopsis.

Malondialdehyde (MDA) is a natural and widespread genotoxin. Given its potentially deleterious effects, it is of interest to establish the identities of the cell types containing this aldehyde. We used in situ chemical trapping with 2-thiobarbituric acid and mass spectrometry with a deuterated standard to characterize MDA pools in the vegetative phase in Arabidopsis thaliana. In leaves, MDA occurred predominantly in the intracellular compartment of mesophyll cells and was enriched in chloroplasts where it was derived primarily from triunsaturated fatty acids (TFAs). High levels of MDA (most of which was unbound) were found within dividing cells in the root tip cell proliferation zone. The bulk of this MDA did not originate from TFAs. We confirmed the localization of MDA in transversal root sections. In addition to MDA in proliferating cells near the root tip we found evidence for the presence of MDA in pericyle cells. Remodeling of non-TFA-derived MDA pools occurred when seedlings were infected with the fungus Botrytis cinerea. Treatment of uninfected seedlings with mediators of plant stress responses (jasmonic acid or salicylic acid) increased seedling MDA levels over 20-fold. In summary, major pools of MDA are associated with cell division foci containing stem cells. The aldehyde is pathogen-inducible in these regions and its levels are increased by cellular mediators that impact defense and growth.

Pathways for the synthesis of polyesters in plants; cutin, suberin, and polyhydroxyalkanoates

Plants naturally produce the lipid-derived polyester cutin, which is found in the plant cuticle that is deposited at the outermost extracellular matrix of the epidermis covering nearly all aboveground tissues. Being at the interface between the cell and the external environment, cutin and the cuticle play important roles in the protection of plants from several stresses. A number of enzymes involved in the synthesis of cutin monomers have recently been identified, including several P450s and one acyl-CoA synthetase, thus representing the first steps toward the understanding of polyester formation and, potentially, polyester engineering to improve the tolerance of plants to stresses, such as drought, and for industrial applications. However, numerous processes underlying cutin synthesis, such as a controlled polymerization, still remain elusive. Suberin is a second polyester found in the extracellular matrix, most often synthesized in root tissues and during secondary growth. Similar to cutin, the function of suberin is to seal off the respective tissue to inhibit water loss and contribute to resistance to pathogen attack. Being the main constituent of cork, suberin is a plant polyester that has already been industrially exploited. Genetic engineering may be worth exploring in order to change the polyester properties for either different applications or to increase cork production in other species. Polyhydroxyalkanoates (PHAs) are attractive polyesters of 3-hydroxyacids because of their properties as bioplastics and elastomers. Although PHAs are naturally found in a wide variety of bacteria, biotechnology has aimed at producing these polymers in plants as a source of cheap and renewable biodegradable plastics. Synthesis of PHA containing various monomers has been demonstrated in the cytosol, plastids, and peroxisomes of plants. Several biochemical pathways have been modified in order to achieve this, including the isoprenoid pathway, the fatty acid biosynthetic pathway, and the fatty acid β-oxidation pathway. PHA synthesis has been demonstrated in a number of plants, including monocots and dicots, and up to 40% PHA per gram dry weight has been demonstrated in Arabidopsis thaliana. Despite some successes, production of PHA in crop plants remains a challenging project. PHA synthesis at high level in vegetative tissues, such as leaves, is associated with chlorosis and reduced growth. The challenge for the future is to succeed in synthesis of PHA copolymers with a narrow range of monomer compositions, at levels that do not compromise plant productivity. This goal will undoubtedly require a deeper understanding of plant biochemical pathways and how carbon fluxes through these pathways can be manipulated, areas where plant "omics" can bring very valuable contributions.

Study of the SRR1-gene family in Arabidopsis, yeast and mouse cells

Abstract: Light is a very important environmental cue for plants. In addition to the energy for photosynthesis, it also provides information that is essential for many processes including seed germination, seedlings development, neighbours detection or transition from the vegetative to the reproductive state. Plants evolved different photoreceptors, among which the phytochromes (PHY), which are red/far-red photoreceptors. This family is composed of 5 members in Arabidopsis thaliana, among which phyB plays the major role for detection of red light. Phytochromes are also able to reset the phase of the circadian clock, which is composed of a complicated network of genes able to produce rhythms of about 24 hours, even in constant conditions. SRR1 (Sensitivity to Red light Reduced) is a gene that was shown to act in the phyB pathway as well as in the circadian clock. It was proposed to play a role in the maintenance of rhythms of the core oscillator because of the circadian phenotype of the srr1 mutant in constant light and in constant darkness. In the present study, we present data confirming the role of SRR1 in the core oscillator. Moreover, we show that SRR1 levels are not limiting for circadian rhythms nor for light perception. We show that the protein levels, the sub-cellular localisation or the complex in which SRR1 is found are not regulated in a circadian manner. Orthologues of SRR1 exist in numerous eukaryotes, forming a new gene family. None of the members of this family have been described. Here, we present data suggesting that the mouse orthologue of SRR1 may not be required for oscillation of the circadian clock of mouse cells in culture. The yeast gene (called BER1 for Benomyl REsistant) was studied to understand the biochemical function of this gene family. Based on synthetic genetic screens, a role of Ber1 was inferred in microtubules dynamics, N-terminal acetylation of protein and proteasome biogenesis. The effect of Ber1 on microtubules was confirmed by the observation that the ber1Δ mutant is more resistant to microtubule-depolymerising drugs and microscopic examination of microtubules in ber 1 Δ mutants. Complementation assays of ber1 Δ mutants and srrl mutants failed to reveal any obvious functional conservation of the mouse, yeast and Arabidopsis orthologues. In conclusion, the SRR1 family might encode genes that either plays different roles in different organisms, or have similar biochemical function but are involved in diverse pathway. Résumé: La lumière est un des facteurs abiotiques les plus important pour les plantes. En plus de l'énergie fournie pour la photosynthèse, elle fourni également de l'information nécessaire pour différents processus comme la germination, le développement des jeunes plantules, la détection de plantes avoisinantes ou encore la transition entre le développement végétatif et reproductif. Plusieurs types de photorécepteurs sont apparus chez les plantes au cours de l'évolution, notamment les phytochromes (PHI, qui perçoivent la lumière rouge et rouge lointaine. Cette famille est composé de 5 membres chez Arabidopsis thaliana, parmi lesquels phyB est le principal récepteur pour la lumière rouge. Les phytochromes sont aussi utiles pour la synchronisation entre les cycles jour-nuit dus à la rotation de la terre et l'horloge circadienne. Cette dernière est composée d'un réseau compliqué qui permet la production de rythmes capables de perdurer même en conditions constantes. SRRI (Sensitivity to Red light Reduced) est un gène qui agit dans la voie de signalisation de phyB ainsi que dans l'horloge circadienne. Il a été proposé que SRRI joue un rôle dans la maintenance des rythmes de l'oscillateur principal à cause des phénotypes circadiens du mutant srrl observés en lumière et en obscurité continue. Dans ce travail, nous présentons des données confirmant le rôle de SRR1 dans l'oscillateur principal. Nous montrons que les niveaux d'expression de SRRI ne sont pas limitants pour les rythmes circadiens ou la perception de la lumière. Enfin, nous montrons que le niveau d'accumulation de la protéine, sa localisation subcellulaire ou encore la taille du complexe dans lequel SRRl est trouvé ne sont pas régulés de façon circadiennes. Des orthologues de SRRI existent chez de nombreux eucaryotes, formant une nouvelle famille de gènes. Aucun des membres de cette famille n'a été étudié avant ce travail. Nous présentons des données suggérant que l'orthologue de la souris n'est peut-être pas requis pour les oscillations de l'horloge circadienne de cellules de souris en culture. Le gène de la levure (appelé SERI pour Benomyl REsistant) a été étudié afin de mieux comprendre la fonction biochimique de cette famille de gène. Une analyse par crible synthétique léthal a révélé un rôle de Ber1 dans la dynamique des microtubules, l'acétylation des protéines en N-terminal et la biogenèse du protéasome. L'effet de Ber1 sur les microtubules a été confirmé par l'observation du mutant ber1 en présence de drogue capable de dépolymériser les microtubules. Celui-ci est plus résistant à ces drogues que le type sauvage. Des expériences de complémentation n'ont pas montré de conservation de la fonction entre SRRI et ses homologues de souris ou de levure. En conclusion, la famille SRRI code pour des gènes qui pourraient avoir soit des rôles différents selon les organismes, soit la même fonction biochimique mais qui serait utile pour des voies de signalisation différentes.

Predictors of awakening from postanoxic status epilepticus after therapeutic hypothermia.

BACKGROUND: Postanoxic status epilepticus (PSE) is considered a predictor of fatal outcome and therefore not intensively treated; however, some patients have had favorable outcomes. The aim of this study was to identify favorable predictors for awakening beyond vegetative state in PSE. METHODS: We studied six subjects treated with hypothermia improving beyond vegetative state after cerebral anoxia, despite PSE. They were among a cohort of patients treated for anoxic encephalopathy with therapeutic hypothermia in our institution between October 1999 and May 2006 (retrospectively, 3/107 patients) and June 2006 and May 2008 (prospectively, 3/74 patients). PSE was defined by clinical and EEG criteria. Outcome was assessed according to the Glasgow-Pittsburgh Cerebral Performance Categories (CPC). RESULTS: All improving patients had preserved brainstem reflexes, cortical somatosensory evoked potentials, and reactive EEG background during PSE. Half of them had myoclonic PSE, while three had nonconvulsive PSE. In the prospective arm, 3/28 patients with PSE showed this clinical-electrophysiologic profile; all awoke. Treatments consisted of benzodiazepines, various antiepileptic drugs, and propofol. One subject died of pneumonia in a minimally conscious state, one patient returned to baseline (CPC1), three had moderate impairment (CPC2), and one remained dependent (CPC3). Patients with nonconvulsive PSE showed a better prognosis than subjects with myoclonic PSE (p = 0.042). CONCLUSION: Patients with postanoxic status epilepticus and preserved brainstem reactions, somatosensory evoked potentials, and EEG reactivity may have a favorable outcome if their condition is treated as status epilepticus.

Metaphyseal chondromatosis combined with D-2-hydroxyglutaric aciduria in four patients.

We report four patients who presented with a severe form of metaphyseal chondromatosis in association with D-2-hydroxyglutaric aciduria (D-2-HGA). All patients showed splaying columns of irregular ossification defects with bulbous metaphyses of the long tubular bones, as well as remarkable involvement of the short tubular and flat bones. The vertebral bodies revealed platyspondyly with irregular, stippled endplates. D-2-HGA has been described as a neurometabolic disorder manifesting a broad range of impairment in mental and motor development. Although hydroxyglutaric acid was excreted in high amounts in the urine of all four patients described herein, no significant neurologic abnormalities were evident. This unusual combination of characteristic skeletal and metabolic abnormalities has rarely been reported. Thus, our report will facilitate the recognition of this distinctive entity, and we suggest that a urine organic acid screening be obtained in patients who present with generalized enchondromatosis.