Reconstitution of water channel function and 2D-crystallization of human aquaporin 8

Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H(2)O(2) in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of three human aquaporins (HsAQP1, HsAQP4, and HsAQP5) have been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP1, HsAQP4 and HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent-lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3?.     

Diversification of lindsaeoid ferns and phylogenetic uncertainty of early polypod relationships

We analysed one nuclear gene (18S) and seven plastid markers [five protein coding (atpA, atpB, rbcL, rpoC1, rps4) and two non‐coding (trnH‐psbA, trnL‐trnF] for 31 members of Polypodiales and four outgroup taxa. We focused our sampling on the lindsaeoids and associated ferns in order to obtain a better understanding of the diversification of the early polypods. However, the exact phylogenetic position of Saccoloma and Cystodium remained uncertain. Based on relaxed molecular clock analyses, it appears that the crown group lindsaeoids diversified in the Caenozoic, more or less simultaneously with the main radiation of other Polypodiales, even though the original divergence between the lindsaeoid and non‐lindsaeoid polypods occurred before the end of the Jurassic. The current pantropical distribution of lindsaeoids can be explained by either long‐distance dispersal across the oceans or vicariance caused by the retreat of previously widely distributed tropical forests from higher to lower latitudes. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 489–503.     

Organ-specific rates of cellular respiration in developing sunflower seedlings and their bearing on metabolic scaling theory

Fifty years ago Max Kleiber described what has become known as the "mouse-to-elephant" curve, i.e., a log-log plot of basal metabolic rate versus body mass. From these data, "Kleiber's 3/4 law" was deduced, which states that metabolic activity scales as the three fourths-power of body mass. However, for reasons unknown so far, no such "universal scaling law" has been discovered for land plants (embryophytes). Here, we report that the metabolic rates of four different organs (cotyledons, cotyledonary hook, hypocotyl, and roots) of developing sunflower (Helianthus annuus L.) seedlings grown in darkness (skotomorphogenesis) and in white light (photomorphogenesis) differ by a factor of 2 to 5 and are largely independent of light treatment. The organ-specific respiration rate (oxygen uptake per minute per gram of fresh mass) of the apical hook, which is composed of cells with densely packaged cytoplasm, is much higher than that of the hypocotyl, an organ that contains vacuolated cells. Data for cell length, cell density, and DNA content reveal that (1) hook opening in white light is caused by a stimulation of cell elongation on the inside of the curved organ, (2) respiration, cell density and DNA content are much higher in the hook than in the stem, and (3) organ-specific respiration rates and the DNA contents of tissues are statistically correlated. We conclude that, due to the heterogeneity of the plant body caused by the vacuolization of the cells, Kleiber's law, which was deduced using mammals as a model system, cannot be applied to embryophytes. In plants, this rule may reflect scaling phenomena at the level of the metabolically active protoplasmic contents of the cells.     

Pharmacokinetics and pharmacodynamics of intravenous artesunate during severe malaria treatment in Ugandan adults

ABSTRACT: BACKGROUND: Severe malaria is a medical emergency with high mortality. Prompt achievement of therapeutic concentrations of highly effective anti-malarial drugs reduces the risk of death. The aim of this study was to assess the pharmacokinetics and pharmacodynamics of intravenous artesunate in Ugandan adults with severe malaria. METHODS: Fourteen adults with severe falciparum malaria requiring parenteral therapy were treated with 2.4 mg/kg intravenous artesunate. Blood samples were collected after the initial dose and plasma concentrations of artesunate and dihydroartemisinin measured by solid-phase extraction and liquid chromatography-tandem mass spectrometry. The study was approved by the Makerere University Faculty of Medicine Research and Ethics Committee (Ref2010-015) and Uganda National Council of Science and Technology (HS605) and registered with ClinicalTrials.gov (NCT01122134). RESULTS: All study participants achieved prompt resolution of symptoms and complete parasite clearance with median (range) parasite clearance time of 17 (8-24) hours. Median (range) maximal artesunate concentration (Cmax) was 3260 (1020-164000) ng/mL, terminal elimination half-life (T1/2) was 0.25 (0.1-1.8) hours and total artesunate exposure (AUC) was 727 (290-111256) ngh/mL. Median (range) dihydroartemisinin Cmax was 3140 (1670-9530) ng/mL, with Tmax of 0.14 (0.6 - 6.07) hours and T1/2 of 1.31 (0.8-2.8) hours. Dihydroartemisinin AUC was 3492 (2183-6338) ngh/mL. None of the participants reported adverse events. CONCLUSIONS: Plasma concentrations of artesunate and dihydroartemisinin were achieved rapidly with rapid and complete symptom resolution and parasite clearance with no adverse events.     

A novel approach to determining violence risk in schizophrenia: developing a stepped strategy in 13,806 discharged patients

Clinical guidelines recommend that violence risk be assessed in schizophrenia. Current approaches are resource-intensive as they employ detailed clinical assessments of dangerousness for most patients. An alternative approach would be to first screen out patients at very low risk of future violence prior to more costly and time-consuming assessments. In order to implement such a stepped strategy, we developed a simple tool to screen out individuals with schizophrenia at very low risk of violent offending. We merged high quality Swedish national registers containing information on psychiatric diagnoses, socio-demographic factors, and violent crime. A cohort of 13,806 individuals with hospital discharge diagnoses of schizophrenia was identified and followed for up to 33 years for violent crime. Cox regression was used to determine risk factors for violent crime and construct the screening tool, the predictive validity of which was measured using four outcome statistics. The instrument was calibrated on 6,903 participants and cross-validated using three independent replication samples of 2,301 participants each. Regression analyses resulted in a tool composed of five items: male sex, previous criminal conviction, young age at assessment, comorbid alcohol abuse, and comorbid drug abuse. At 5 years after discharge, the instrument had a negative predictive value of 0.99 (95% CI = 0.98–0.99), meaning that very few individuals who the tool screened out (n = 2,359 out of original sample of 6,903) were subsequently convicted of a violent offence. Screening out patients who are at very low risk of violence prior to more detailed clinical assessment may assist the risk assessment process in schizophrenia.     

Climatic niche shift predicts thermal trait response in one but not both introductions of the Puerto Rican lizard Anolis cristatellus to Miami, Florida, USA

Global change is predicted to alter environmental conditions for populations in numerous ways; for example, invasive species often experience substantial shifts in climatic conditions during introduction from their native to non-native ranges. Whether these shifts elicit a phenotypic response, and how adaptation and phenotypic plasticity contribute to phenotypic change, are key issues for understanding biological invasions and how populations may respond to local climate change. We combined modeling, field data, and a laboratory experiment to test for changing thermal tolerances during the introduction of the tropical lizard Anolis cristatellus from Puerto Rico to Miami, Florida. Species distribution models and bioclimatic data analyses showed lower minimum temperatures, and greater seasonal and annual variation in temperature for Miami compared to Puerto Rico. Two separate introductions of A. cristatellus occurred in Miami about 12 km apart, one in South Miami and the other on Key Biscayne, an offshore island. As predicted from the shift in the thermal climate and the thermal tolerances of other Anolis species in Miami, laboratory acclimation and field acclimatization showed that the introduced South Miami population of A. cristatellus has diverged from its native-range source population by acquiring low-temperature acclimation ability. By contrast, the introduced Key Biscayne population showed little change compared to its source. Our analyses predicted an adaptive response for introduced populations, but our comparisons to native-range sources provided evidence for thermal plasticity in one introduced population but not the other. The rapid acquisition of thermal plasticity by A. cristatellus in South Miami may be advantageous for its long-term persistence there and expansion of its non-native range. Our results also suggest that the common assumption of no trait variation when modeling non-native species distributions is invalid.     

Metabolic flux rearrangement in the amino acid metabolism reduces ammonia stress in the α1-antitrypsin producing human AGE1.HN cell line

This study focused on metabolic changes in the neuronal human cell line AGE1.HN upon increased ammonia stress. Batch cultivations of α(1)-antitrypsin (A1AT) producing AGE1.HN cells were carried out in media with initial ammonia concentrations ranging from 0mM to 5mM. Growth, A1AT production, metabolite dynamics and finally metabolic fluxes calculated by metabolite balancing were compared. Growth and A1AT production decreased with increasing ammonia concentration. The maximum A1AT concentration decreased from 0.63g/l to 0.51g/l. Central energy metabolism remained relatively unaffected exhibiting only slightly increased glycolytic flux at high initial ammonia concentration in the medium. However, the amino acid metabolism was significantly changed. Fluxes through transaminases involved in amino acid degradation were reduced concurrently with a reduced uptake of amino acids. On the other hand fluxes through transaminases working in the direction of amino acid synthesis, i.e., alanine and phosphoserine, were increased leading to increased storage of excess nitrogen in extracellular alanine and serine. Glutamate dehydrogenase flux was reversed increasingly fixing free ammonia with increasing ammonia concentration. Urea production additionally observed was associated with arginine uptake by the cells and did not increase at high ammonia stress. It was therefore not used as nitrogen sink to remove excess ammonia. The results indicate that the AGE1.HN cell line can adapt to ammonia concentrations usually present during the cultivation process to a large extent by changing metabolism but with slightly reduced A1AT production and growth.     

Peptidome Analysis of Cerebrospinal Fluid by LC-MALDI MS

We report on the analysis of endogenous peptides in cerebrospinal fluid (CSF) by mass spectrometry. A method was developed for preparation of peptide extracts from CSF. Analysis of the extracts by offline LC-MALDI MS resulted in the detection of 3,000-4,000 peptide-like features. Out of these, 730 peptides were identified by MS/MS. The majority of these peptides have not been previously reported in CSF. The identified peptides were found to originate from 104 proteins, of which several have been reported to be involved in different disorders of the central nervous system. These results support the notion that CSF peptidomics may be viable complement to proteomics in the search of biomarkers of CNS disorders.     

Sickness Absence following Coronary Revascularisation. A National Study of Women and Men of Working Age in Sweden 1994-2006

Background

Evidence based and gender specific knowledge about sickness absence following coronary revascularisation is lacking. The objective was to investigate sickness absence after a first coronary artery bypass grafting (CABG) or percutaneous coronary intervention (PCI) among women and men in a national Swedish study.

Materials and Methods

All patients 30–63 years of age, who underwent a first CABG (n = 22,985, 16% women) or PCI (40,891, 22% women) in Sweden between 1994 and 2006 were included. Information on sickness absence, co-morbidity, and other patient characteristics was obtained from national registers. Long-term sickness absence (LTSA) was defined as >180 and >90 sick-leave days in the first sick-leave spell following CABG and PCI, respectively. Prevalence ratio (PR) and 95% confidence interval (CI) of LTSA were calculated.

Findings

LTSA followed the interventions in 41% and 36% for CABG and PCI patients, respectively. Women had more often LTSA compared with men, (CABG PR = 1.23: 95% CI 1.19–1.28 and PCI PR = 1.19; 95% CI 1.16–1.23). A history of sickness absence the year before the intervention increased the risk for LTSA after the intervention in both genders. Among women, older age, or being self employed or unemployed was associated with a lower risk for LTSA. Among men previous cardiovascular disease, diabetes and low socio-economic position increased the risk. During the observation period, there was no change in sickness absence rates among PCI patients but an increase among CABG patients adjusting for patient characteristics.

Conclusion

This national study covering a 13-year period shows that long-term sickness absence following coronary revascularisation is common in Sweden, especially among women, and is associated with socio-economic position, co-morbidity, and sickness absence during the year before the intervention. Gender specific scientific knowledge about use and effects of sickness absence following coronary revascularisation is warranted for the patients, the treating physicians, the healthcare sector, and the society.